ALBERT

Description: Some of the most dangerous pathogens such as Mycobacterium tuberculosis and Yersinia pestis evolve clonally . This means that little or no recombination occurs between strains belonging to these species. Paradoxically, although different members of these species show extreme sequence similarity of orthologous genes, some show considerable intraspecies phenotypic variation, the source of which remains elusive. To examine the possible sources of phenotypic variation within clonal pathogenic bacterial species, we carried out an extensive genomic and pan-genomic analysis of the sources of genetic variation available to a large collection of clonal and nonclonal pathogenic bacterial species. We show that while nonclonal species diversify through a combination of changes to gene sequences, gene loss and gene gain, gene loss completely dominates as a source of genetic variation within clonal species. Indeed, gene loss is so prevalent within clonal species as to lead to levels of gene content variation comparable to those found in some nonclonal species that are much more diverged in their gene sequences and that acquire a substantial number of genes horizontally. Gene loss therefore needs to be taken into account as a potential dominant source of phenotypic variation within clonal bacterial species.

Description: Obligate bacterial symbionts are widespread in many invertebrates, where they are often confined to specialized host cells and are transmitted directly from mother to progeny. Increasing numbers of these bacteria are being characterized but questions remain about their population structure and evolution. Here we take a comparative genomics approach to investigate two prominent bacterial symbionts (BFo1 and BFo2) isolated from geographically separated populations of western flower thrips, Frankliniella occidentalis. Our multifaceted approach to classifying these symbionts includes concatenated multilocus sequence analysis (MLSA) phylogenies, ribosomal multilocus sequence typing (rMLST), construction of whole-genome phylogenies, and in-depth genomic comparisons. We showed that the BFo1 genome clusters more closely to species in the genus Erwinia, and is a putative close relative to Erwinia aphidicola . BFo1 is also likely to have shared a common ancestor with Erwinia pyrifoliae/Erwinia amylovora and the nonpathogenic Erwinia tasmaniensis and genetic traits similar to Erwinia billingiae . The BFo1 genome contained virulence factors found in the genus Erwinia but represented a divergent lineage. In contrast, we showed that BFo2 belongs within the Enterobacteriales but does not group closely with any currently known bacterial species. Concatenated MLSA phylogenies indicate that it may have shared a common ancestor to the Erwinia and Pantoea genera, and based on the clustering of rMLST genes, it was most closely related to Pantoea ananatis but represented a divergent lineage. We reconstructed a core genome of a putative common ancestor of Erwinia and Pantoea and compared this with the genomes of BFo bacteria. BFo2 possessed none of the virulence determinants that were omnipresent in the Erwinia and Pantoea genera. Taken together, these data are consistent with BFo2 representing a highly novel species that maybe related to known Pantoea .

Description: Gene expression evolution occurs through changes in cis - or trans -regulatory elements or both. Interactions between transcription factors (TFs) and their binding sites (TFBSs) constitute one of the most important points where these two regulatory components intersect. In this study, we investigated the evolution of TFBSs in the promoter regions of different Saccharomyces strains and species. We divided the promoter of a gene into the proximal region and the distal region, which are defined, respectively, as the 200-bp region upstream of the transcription starting site and as the 200-bp region upstream of the proximal region. We found that the predicted TFBSs in the proximal promoter regions tend to be evolutionarily more conserved than those in the distal promoter regions. Additionally, Saccharomyces cerevisiae strains used in the fermentation of alcoholic drinks have experienced more TFBS losses than gains compared with strains from other environments (wild strains, laboratory strains, and clinical strains). We also showed that differences in TFBSs correlate with the cis component of gene expression evolution between species (comparing S. cerevisiae and its sister species Saccharomyces paradoxus ) and within species (comparing two closely related S. cerevisiae strains).

Description: Gene duplication is a key factor contributing to phenotype diversity across and within species. Although the availability of complete genomes has led to the extensive study of genomic duplications, the dynamics and variability of gene duplications mediated by retrotransposition are not well understood. Here, we predict mRNA retrotransposition and use comparative genomics to investigate their origin and variability across primates. Analyzing seven anthropoid primate genomes, we found a similar number of mRNA retrotranspositions (~7,500 retrocopies) in Catarrhini (Old Word Monkeys, including humans), but a surprising large number of retrocopies (~10,000) in Platyrrhini (New World Monkeys), which may be a by-product of higher long interspersed nuclear element 1 activity in these genomes. By inferring retrocopy orthology, we dated most of the primate retrocopy origins, and estimated a decrease in the fixation rate in recent primate history, implying a smaller number of species-specific retrocopies. Moreover, using RNA-Seq data, we identified approximately 3,600 expressed retrocopies. As expected, most of these retrocopies are located near or within known genes, present tissue-specific and even species-specific expression patterns, and no expression correlation to their parental genes. Taken together, our results provide further evidence that mRNA retrotransposition is an active mechanism in primate evolution and suggest that retrocopies may not only introduce great genetic variability between lineages but also create a large reservoir of potentially functional new genomic loci in primate genomes.

Description: Viruses rely completely on the hosts’ machinery for translation of viral transcripts. However, for most viruses infecting humans, codon usage preferences (CUPrefs) do not match those of the host. Human papillomaviruses (HPVs) are a showcase to tackle this paradox: they present a large genotypic diversity and a broad range of phenotypic presentations, from asymptomatic infections to productive lesions and cancer. By applying phylogenetic inference and dimensionality reduction methods, we demonstrate first that genes in HPVs are poorly adapted to the average human CUPrefs, the only exception being capsid genes in viruses causing productive lesions. Phylogenetic relationships between HPVs explained only a small proportion of CUPrefs variation. Instead, the most important explanatory factor for viral CUPrefs was infection phenotype, as orthologous genes in viruses with similar clinical presentation displayed similar CUPrefs. Moreover, viral genes with similar spatiotemporal expression patterns also showed similar CUPrefs. Our results suggest that CUPrefs in HPVs reflect either variations in the mutation bias or differential selection pressures depending on the clinical presentation and expression timing. We propose that poor viral CUPrefs may be central to a trade-off between strong viral gene expression and the potential for eliciting protective immune response.

Description: Historically, genome-wide and molecular characterization of the genus Listeria has concentrated on the important human pathogen Listeria monocytogenes and a small number of closely related species, together termed Listeria sensu strictu. More recently, a number of genome sequences for more basal, and nonpathogenic, members of the Listeria genus have become available, facilitating a wider perspective on the evolution of pathogenicity and genome level evolutionary dynamics within the entire genus (termed Listeria sensu lato). Here, we have sequenced the genomes of additional Listeria fleischmannii and Listeria newyorkensis isolates and explored the dynamics of genome evolution in Listeria sensu lato. Our analyses suggest that acquisition of genetic material through gene duplication and divergence as well as through lateral gene transfer (mostly from outside Listeria ) is widespread throughout the genus. Novel genetic material is apparently subject to rapid turnover. Multiple lines of evidence point to significant differences in evolutionary dynamics between the most basal Listeria subclade and all other congeners, including both sensu strictu and other sensu lato isolates. Strikingly, these differences are likely attributable to stochastic, population-level processes and contribute to observed variation in genome size across the genus. Notably, our analyses indicate that the common ancestor of Listeria sensu lato lacked flagella, which were acquired by lateral gene transfer by a common ancestor of Listeria grayi and Listeria sensu strictu, whereas a recently functionally characterized pathogenicity island, responsible for the capacity to produce cobalamin and utilize ethanolamine/propane-2-diol, was acquired in an ancestor of Listeria sensu strictu.

Description: Taeniid cestodes (including the human parasites Echinococcus spp. and Taenia solium ) have very few mobile genetic elements (MGEs) in their genome, despite lacking a canonical PIWI pathway. The MGEs of these parasites are virtually unexplored, and nothing is known about their expression and silencing. In this work, we report the discovery of a novel family of small nonautonomous long terminal repeat retrotransposons (also known as terminal-repeat retrotransposons in miniature, TRIMs) which we have named ta-TRIM (taeniid TRIM). ta-TRIM s are only the second family of TRIM elements discovered in animals, and are likely the result of convergent reductive evolution in different taxonomic groups. These elements originated at the base of the taeniid tree and have expanded during taeniid diversification, including after the divergence of closely related species such as Echinococcus multilocularis and Echinococcus granulosus . They are massively expressed in larval stages, from a small proportion of full-length copies and from isolated terminal repeats that show transcriptional read-through into downstream regions, generating novel noncoding RNAs and transcriptional fusions to coding genes. In E. multilocularis , ta-TRIM s are specifically expressed in the germinative cells (the somatic stem cells) during asexual reproduction of metacestode larvae. This would provide a developmental mechanism for insertion of ta-TRIM s into cells that will eventually generate the adult germ line. Future studies of active and inactive ta-TRIM elements could give the first clues on MGE silencing mechanisms in cestodes.

Description: Even though mitochondrial genomes, which characterize eukaryotic cells, were first discovered more than 50 years ago, mitochondrial genomics remains an important topic in molecular biology and genome sciences. The Phylum Alveolata comprises three major groups (ciliates, apicomplexans, and dinoflagellates), the mitochondrial genomes of which have diverged widely. Even though the gene content of dinoflagellate mitochondrial genomes is reportedly comparable to that of apicomplexans, the highly fragmented and rearranged genome structures of dinoflagellates have frustrated whole genomic analysis. Consequently, noncoding sequences and gene arrangements of dinoflagellate mitochondrial genomes have not been well characterized. Here we report that the continuous assembled genome (~326 kb) of the dinoflagellate, Symbiodinium minutum , is AT-rich (~64.3%) and that it contains three protein-coding genes. Based upon in silico analysis, the remaining 99% of the genome comprises transcriptomic noncoding sequences. RNA edited sites and unique, possible start and stop codons clarify conserved regions among dinoflagellates. Our massive transcriptome analysis shows that almost all regions of the genome are transcribed, including 27 possible fragmented ribosomal RNA genes and 12 uncharacterized small RNAs that are similar to mitochondrial RNA genes of the malarial parasite, Plasmodium falciparum . Gene map comparisons show that gene order is only slightly conserved between S. minutu m and P. falciparum . However, small RNAs and intergenic sequences share sequence similarities with P. falciparum , suggesting that the function of noncoding sequences has been preserved despite development of very different genome structures.

Description: Evolutionary studies usually use a two-step process to investigate sequence data. Step one estimates a multiple sequence alignment (MSA) and step two applies phylogenetic methods to ask evolutionary questions of that MSA. Modern phylogenetic methods infer evolutionary parameters using maximum likelihood or Bayesian inference, mediated by a probabilistic substitution model that describes sequence change over a tree. The statistical properties of these methods mean that more data directly translates to an increased confidence in downstream results, providing the substitution model is adequate and the MSA is correct. Many studies have investigated the robustness of phylogenetic methods in the presence of substitution model misspecification, but few have examined the statistical properties of those methods when the MSA is unknown. This simulation study examines the statistical properties of the complete two-step process when inferring sequence divergence and the phylogenetic tree topology. Both nucleotide and amino acid analyses are negatively affected by the alignment step, both through inaccurate guide tree estimates and through overfitting to that guide tree. For many alignment tools these effects become more pronounced when additional sequences are added to the analysis. Nucleotide sequences are particularly susceptible, with MSA errors leading to statistical support for long-branch attraction artifacts, which are usually associated with gross substitution model misspecification. Amino acid MSAs are more robust, but do tend to arbitrarily resolve multifurcations in favor of the guide tree. No inference strategies produce consistently accurate estimates of divergence between sequences, although amino acid MSAs are again more accurate than their nucleotide counterparts. We conclude with some practical suggestions about how to limit the effect of MSA uncertainty on evolutionary inference.

Description: The evolution of mitochondrial information processing pathways, including replication, transcription and translation, is characterized by the gradual replacement of mitochondrial-encoded proteins with nuclear-encoded counterparts of diverse evolutionary origins. Although the ancestral enzymes involved in mitochondrial transcription and replication have been replaced early in eukaryotic evolution, mitochondrial translation is still carried out by an apparatus largely inherited from the α-proteobacterial ancestor. However, variation in the complement of mitochondrial-encoded molecules involved in translation, including transfer RNAs (tRNAs), provides evidence for the ongoing evolution of mitochondrial protein synthesis. Here, we investigate the evolution of the mitochondrial translational machinery using recent genomic and transcriptomic data from animals that have experienced the loss of mt-tRNAs, including phyla Cnidaria and Ctenophora, as well as some representatives of all four classes of Porifera. We focus on four sets of mitochondrial enzymes that directly interact with tRNAs: Aminoacyl-tRNA synthetases, glutamyl-tRNA amidotransferase, tRNA Ile lysidine synthetase, and RNase P. Our results support the observation that the fate of nuclear-encoded mitochondrial proteins is influenced by the evolution of molecules encoded in mitochondrial DNA, but in a more complex manner than appreciated previously. The data also suggest that relaxed selection on mitochondrial translation rather than coevolution between mitochondrial and nuclear subunits is responsible for elevated rates of evolution in mitochondrial translational proteins.

Description: The expansion of DUF1220 domain copy number during human evolution is a dramatic example of rapid and repeated domain duplication. Although patterns of expression, homology, and disease associations suggest a role in cortical development, this hypothesis has not been robustly tested using phylogenetic methods. Here, we estimate DUF1220 domain counts across 12 primate genomes using a nucleotide Hidden Markov Model. We then test a series of hypotheses designed to examine the potential evolutionary significance of DUF1220 copy number expansion. Our results suggest a robust association with brain size, and more specifically neocortex volume. In contradiction to previous hypotheses, we find a strong association with postnatal brain development but not with prenatal brain development. Our results provide further evidence of a conserved association between specific loci and brain size across primates, suggesting that human brain evolution may have occurred through a continuation of existing processes.

Description: Alternative splicing and gene duplication are the two main processes responsible for expanding protein functional diversity. Although gene duplication can generate new genes and alternative splicing can introduce variation through alternative gene products, the interplay between the two processes is complex and poorly understood. Here, we have carried out a study of the evolution of alternatively spliced exons after gene duplication to better understand the interaction between the two processes. We created a manually curated set of 97 human genes with mutually exclusively spliced homologous exons and analyzed the evolution of these exons across five distantly related vertebrates (lamprey, spotted gar, zebrafish, fugu, and coelacanth). Most of these exons had an ancient origin (more than 400 Ma). We found examples supporting two extreme evolutionary models for the behaviour of homologous axons after gene duplication. We observed 11 events in which gene duplication was accompanied by splice isoform separation, that is, each paralog specifically conserved just one distinct ancestral homologous exon. At other extreme, we identified genes in which the homologous exons were always conserved within paralogs, suggesting that the alternative splicing event cannot easily be separated from the function in these genes. That many homologous exons fall in between these two extremes highlights the diversity of biological systems and suggests that the subtle balance between alternative splicing and gene duplication is adjusted to the specific cellular context of each gene.

Description: There is widespread interest today in understanding enhancers, which are regulatory elements typically harboring several transcription factor binding sites and mediating the combinatorial effect of transcription factors on gene expression. The evolution of enhancers poses interesting unanswered questions, for example, the evolutionary time taken for a typical enhancer to emerge or the factors shaping its evolution. Existing approaches to cis -regulatory evolution have often ignored the combinatorial nature and varied biochemical mechanisms of gene regulation encoded in enhancers. We report on our investigation of enhancer evolution through the use of PEBCRES, a framework for evolutionary simulation of enhancers that employs a mechanistic and well-supported sequence-to-expression model to assign fitness to the evolving enhancer genotype. We estimated the time necessary to evolve, from genomic background, enhancers capable of driving complex gene expression patterns similar to those involved in early development in Drosophila. We found the time-to-evolve to range between 0.5 and 10 Myr, and to vary greatly with the target expression pattern, complexity of the real enhancer known to encode that pattern, and the strength of input from specific transcription factors. To our knowledge, this is the first estimate of waiting times for realistic enhancers to evolve. The in silico evolved enhancers had, with a few interesting exceptions, site compositions similar to those seen in real enhancers for the same patterns. Our simulations also revealed that certain features of an enhancer might evolve not due to their biological function but as aids to the evolutionary process itself.

Description: Despite significant progress in the structural and functional characterization of the human genome, understanding of the mechanisms underlying the genetic basis of human phenotypic uniqueness remains limited. Here, I report that transposable element-derived sequences, most notably LTR7/HERV-H, LTR5_Hs, and L1HS, harbor 99.8% of the candidate human-specific regulatory loci (HSRL) with putative transcription factor-binding sites in the genome of human embryonic stem cells (hESC). A total of 4,094 candidate HSRL display selective and site-specific binding of critical regulators (NANOG [Nanog homeobox], POU5F1 [POU class 5 homeobox 1], CCCTC-binding factor [CTCF], Lamin B1), and are preferentially located within the matrix of transcriptionally active DNA segments that are hypermethylated in hESC. hESC-specific NANOG-binding sites are enriched near the protein-coding genes regulating brain size, pluripotency long noncoding RNAs, hESC enhancers, and 5-hydroxymethylcytosine-harboring regions immediately adjacent to binding sites. Sequences of only 4.3% of hESC-specific NANOG-binding sites are present in Neanderthals’ genome, suggesting that a majority of these regulatory elements emerged in Modern Humans. Comparisons of estimated creation rates of novel TF-binding sites revealed that there was 49.7-fold acceleration of creation rates of NANOG-binding sites in genomes of Chimpanzees compared with the mouse genomes and further 5.7-fold acceleration in genomes of Modern Humans compared with the Chimpanzees genomes. Preliminary estimates suggest that emergence of one novel NANOG-binding site detectable in hESC required 466 years of evolution. Pathway analysis of coding genes that have hESC-specific NANOG-binding sites within gene bodies or near gene boundaries revealed their association with physiological development and functions of nervous and cardiovascular systems, embryonic development, behavior, as well as development of a diverse spectrum of pathological conditions such as cancer, diseases of cardiovascular and reproductive systems, metabolic diseases, multiple neurological and psychological disorders. A proximity placement model is proposed explaining how a 33–47% excess of NANOG, CTCF, and POU5F1 proteins immobilized on a DNA scaffold may play a functional role at distal regulatory elements.

Description: Organisms can adapt to local environmental conditions as a plastic response or become adapted through natural selection on genetic variation. The ability to adapt to increased water temperatures will be of paramount importance for many fish species as the climate continues to warm and water resources become limited. Because increased water temperatures will reduce the dissolved oxygen available for fish, we hypothesized that adaptation to low oxygen environments would involve improved respiration through oxidative phosphorylation (OXPHOS). To test this hypothesis, we subjected individuals from two ecologically divergent populations of inland (redband) rainbow trout ( Oncorhynchus mykiss gairdneri ) with historically different temperature regimes (desert and montane) and their F1 progeny to diel cycles of temperature stress and then examined gene expression data for 80 nuclear- and mitochondrial-encoded OXPHOS subunits that participate in respiration. Of the 80 transcripts, 7 showed ≥ 2-fold difference in expression levels in gill tissue from desert fish under heat stress whereas the montane fish had none and the F1 only had one differentially expressed gene. A structural analysis of the proteins encoded by those genes suggests that the response could coordinate the formation of supercomplexes and oligomers. Supercomplexes may increase the efficiency of respiration because complexes I, III, and IV are brought into close proximity and oligomerization of complex V alters the macrostructure of mitochondria to improve respiration. Significant differences in gene expression patterns in response to heat stress in a common environment indicate that the response was not due to plasticity but had a genetic basis.

Description: The enigmatic monocot family Triuridaceae provides a potentially useful model system for studying the effects of an ancient loss of photosynthesis on the plant plastid genome, as all of its members are mycoheterotrophic and achlorophyllous. However, few studies have placed the family in a comparative context, and its phylogenetic placement is only partly resolved. It was also unclear whether any taxa in this family have retained a plastid genome. Here, we used genome survey sequencing to retrieve plastid genome data for Sciaphila densiflora (Triuridaceae) and ten autotrophic relatives in the orders Dioscoreales and Pandanales. We recovered a highly reduced plastome for Sciaphila that is nearly colinear with Carludovica palmata , a photosynthetic relative that belongs to its sister group in Pandanales, Cyclanthaceae–Pandanaceae. This phylogenetic placement is well supported and robust to a broad range of analytical assumptions in maximum-likelihood inference, and is congruent with recent findings based on nuclear and mitochondrial evidence. The 28 genes retained in the S. densiflora plastid genome are involved in translation and other nonphotosynthetic functions, and we demonstrate that nearly all of the 18 protein-coding genes are under strong purifying selection. Our study confirms the utility of whole plastid genome data in phylogenetic studies of highly modified heterotrophic plants, even when they have substantially elevated rates of substitution.

Description: We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis . These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling.

Description: Most sequenced eukaryotic genomes show a large excess of recent duplicates. As duplicates age, both the population genetic process of failed fixation and the mutation-driven process of nonfunctionalization act to reduce the observed number of duplicates. Understanding the processes generating the age distributions of recent duplicates is important to also understand the role of duplicate genes in the functional divergence of genomes. To date, mechanistic models for duplicate gene retention only account for the mutation-driven nonfunctionalization process. Here, a neutral model for the distribution of synonymous substitutions in duplicated genes which are segregating and expected to never fix in a population is introduced. This model enables differentiation of neutral loss due to failed fixation from loss due to mutation-driven nonfunctionalization. The model has been validated on simulated data and subsequent analysis with the model on genomic data from human and mouse shows that conclusions about the underlying mechanisms for duplicate gene retention can be sensitive to consideration of population genetic processes.

Description: Stenotrophomonas maltophilia , a ubiquitous Gram-negative -proteobacterium, has emerged as an important opportunistic pathogen responsible for nosocomial infections. A major characteristic of clinical isolates is their high intrinsic or acquired antibiotic resistance level. The aim of this study was to decipher the genetic determinism of antibiotic resistance among strains from different origins (i.e., natural environment and clinical origin) showing various antibiotic resistance profiles. To this purpose, we selected three strains isolated from soil collected in France or Burkina Faso that showed contrasting antibiotic resistance profiles. After whole-genome sequencing, the phylogenetic relationships of these 3 strains and 11 strains with available genome sequences were determined. Results showed that a strain’s phylogeny did not match their origin or antibiotic resistance profiles. Numerous antibiotic resistance coding genes and efflux pump operons were revealed by the genome analysis, with 57% of the identified genes not previously described. No major variation in the antibiotic resistance gene content was observed between strains irrespective of their origin and antibiotic resistance profiles. Although environmental strains generally carry as many multidrug resistant (MDR) efflux pumps as clinical strains, the absence of resistance–nodulation–division (RND) pumps (i.e., SmeABC) previously described to be specific to S. maltophilia was revealed in two environmental strains (BurA1 and PierC1). Furthermore the genome analysis of the environmental MDR strain BurA1 showed the absence of SmeABC but the presence of another putative MDR RND efflux pump, named EbyCAB on a genomic island probably acquired through horizontal gene transfer.

Description: Nucleobase modifications dramatically alter nucleic acid structure and thermodynamics. 2-thiouridine (s 2 U) is a modified nucleobase found in tRNAs and known to stabilize U:A base pairs and destabilize U:G wobble pairs. The recently reported crystal structures of s 2 U-containing RNA duplexes do not entirely explain the mechanisms responsible for the stabilizing effect of s 2 U or whether this effect is entropic or enthalpic in origin. We present here thermodynamic evaluations of duplex formation using ITC and UV thermal denaturation with RNA duplexes containing internal s 2 U:A and s 2 U:U pairs and their native counterparts. These results indicate that s 2 U stabilizes both duplexes. The stabilizing effect is entropic in origin and likely results from the s 2 U-induced preorganization of the single-stranded RNA prior to hybridization. The same preorganizing effect is likely responsible for structurally resolving the s 2 U:U pair-containing duplex into a single conformation with a well-defined H-bond geometry. We also evaluate the effect of s 2 U on single strand conformation using UV- and CD-monitored thermal denaturation and on nucleoside conformation using 1 H NMR spectroscopy, MD and umbrella sampling. These results provide insights into the effects that nucleobase modification has on RNA structure and thermodynamics and inform efforts toward improving both ribozyme-catalyzed and nonenzymatic RNA copying.

Description: DNA interstrand crosslinks (ICLs) are the primary mechanism for the cytotoxic activity of many clinical anticancer drugs, and numerous strategies for forming ICLs have been developed. One such method is using crosslink-forming oligonucleotides (CFOs). In this study, we designed a 4-amino-6-oxo-2-vinylpyrimidine (AOVP) derivative with an acyclic spacer to react selectively with guanine. The AOVP CFO exhibited selective crosslinking reactivity with guanine and thymine in DNA, and with guanine in RNA. These crosslinking reactions with guanine were accelerated in the presence of CoCl 2 , NiCl 2 , ZnCl 2 and MnCl 2 . In addition, we demonstrated that the AOVP CFO was reactive toward 8-oxoguanine opposite AOVP in the duplex DNA. The structural analysis of each guanine and 8-oxoguanine adduct in the duplex DNA was investigated by high-resolution NMR. The results suggested that AOVP reacts at the N2 amine in guanine and at the N1 or N2 amines in 8-oxoguanine in the duplex DNA. This study demonstrated the first direct determination of the adduct structure in duplex DNA without enzyme digestion.

Description: The sequencing of the full transcriptome (RNA-seq) has become the preferred choice for the measurement of genome-wide gene expression. Despite its widespread use, challenges remain in RNA-seq data analysis. One often-overlooked aspect is normalization. Despite the fact that a variety of factors or ‘batch effects’ can contribute unwanted variation to the data, commonly used RNA-seq normalization methods only correct for sequencing depth. The study of gene expression is particularly problematic when it is influenced simultaneously by a variety of biological factors in addition to the one of interest. Using examples from experimental neuroscience, we show that batch effects can dominate the signal of interest; and that the choice of normalization method affects the power and reproducibility of the results. While commonly used global normalization methods are not able to adequately normalize the data, more recently developed RNA-seq normalization can. We focus on one particular method, RUVSeq and show that it is able to increase power and biological insight of the results. Finally, we provide a tutorial outlining the implementation of RUVSeq normalization that is applicable to a broad range of studies as well as meta-analysis of publicly available data.

Description: Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13.

Description: A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea.

Description: Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson–Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine–thymine (A–T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events.

Description: Sequence alignment is a long standing problem in bioinformatics. The Basic Local Alignment Search Tool (BLAST) is one of the most popular and fundamental alignment tools. The explosive growth of biological sequences calls for speedup of sequence alignment tools such as BLAST. To this end, we develop high speed BLASTN (HS-BLASTN), a parallel and fast nucleotide database search tool that accelerates MegaBLAST—the default module of NCBI-BLASTN. HS-BLASTN builds a new lookup table using the FMD-index of the database and employs an accurate and effective seeding method to find short stretches of identities (called seeds) between the query and the database. HS-BLASTN produces the same alignment results as MegaBLAST and its computational speed is much faster than MegaBLAST. Specifically, our experiments conducted on a 12-core server show that HS-BLASTN can be 22 times faster than MegaBLAST and exhibits better parallel performance than MegaBLAST. HS-BLASTN is written in C++ and the related source code is available at https://github.com/chenying2016/queries under the GPLv3 license.

Description: Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo . Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors.

Description: Ten eleven translocation (Tet) family-mediated DNA oxidation on 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) represents a novel epigenetic modification that regulates dynamic gene expression during embryonic stem cells (ESCs) differentiation. Through the role of Tet on 5hmC regulation in stem cell development is relatively defined, how the Tet family is regulated and impacts on ESCs lineage development remains elusive. In this study, we show non-coding RNA regulation on Tet family may contribute to epigenetic regulation during ESCs differentiation, which is suggested by microRNA-29b (miR-29b) binding sites on the Tet1 3' untranslated region (3' UTR). We demonstrate miR-29b increases sharply after embyoid body (EB) formation, which causes Tet1 repression and reduction of cellular 5hmC level during ESCs differentiation. Importantly, we show this miR-29b/Tet1 regulatory axis promotes the mesendoderm lineage formation both in vitro and in vivo by inducing the Nodal signaling pathway and repressing the key target of the active demethylation pathway, Tdg. Taken together, our findings underscore the contribution of small non-coding RNA mediated regulation on DNA demethylation dynamics and the differential expressions of key mesendoderm regulators during ESCs lineage specification. MiR-29b could potentially be applied to enrich production of mesoderm and endoderm derivatives and be further differentiated into desired organ-specific cells.

Description: The emergence of multidrug-resistant pathogens will make current antibiotics ineffective. For linezolid, a member of the novel oxazolidinone class of antibiotics, 10 nucleotide mutations in the ribosome have been described conferring resistance. Hypotheses for how these mutations affect antibiotics binding have been derived based on comparative crystallographic studies. However, a detailed description at the atomistic level of how remote mutations exert long-distance effects has remained elusive. Here, we show that the G2032A-C2499A double mutation, located 〉 10 Å away from the antibiotic, confers linezolid resistance by a complex set of effects that percolate to the binding site. By molecular dynamics simulations and free energy calculations, we identify U2504 and C2452 as spearheads among binding site nucleotides that exert the most immediate effect on linezolid binding. Structural reorganizations within the ribosomal subunit due to the mutations are likely associated with mutually compensating changes in the effective energy. Furthermore, we suggest two main routes of information transfer from the mutation sites to U2504 and C2452. Between these, we observe cross-talk, which suggests that synergistic effects observed for the two mutations arise in an indirect manner. These results should be relevant for the development of oxazolidinone derivatives that are active against linezolid-resistant strains.

Description: Genes from yeast to mammals are frequently subject to non-coding transcription of their antisense strand; however the genome-wide role for antisense transcription remains elusive. As transcription influences chromatin structure, we took a genome-wide approach to assess which chromatin features are associated with nascent antisense transcription, and contrast these with features associated with nascent sense transcription. We describe a distinct chromatin architecture at the promoter and gene body specifically associated with antisense transcription, marked by reduced H2B ubiquitination, H3K36 and H3K79 trimethylation and increased levels of H3 acetylation, chromatin remodelling enzymes, histone chaperones and histone turnover. The difference in sense transcription between genes with high or low levels of antisense transcription is slight; thus the antisense transcription-associated chromatin state is not simply analogous to a repressed state. Using mutants in which the level of antisense transcription is reduced at GAL1 , or altered genome-wide, we show that non-coding transcription is associated with high H3 acetylation and H3 levels across the gene, while reducing H3K36me3. Set1 is required for these antisense transcription-associated chromatin changes in the gene body. We propose that nascent antisense and sense transcription have fundamentally distinct relationships with chromatin, and that both should be considered canonical features of eukaryotic genes.

Description: The expansion of Bantu-speaking agropastoralist populations had a great impact on the genetic, linguistic, and cultural variation of sub-Saharan Africa. It is generally accepted that Bantu languages originated in an area around the present border between Cameroon and Nigeria approximately 5,000 years ago, from where they spread South and East becoming the largest African linguistic branch. The demic consequences of this event are reflected in the relatively high genetic homogeneity observed across most of sub-Saharan Africa populations. In this work, we explored genome-wide single nucleotide polymorphism data from 28 populations to characterize the genetic components present in sub-Saharan African populations. Combining novel data from four Southern African populations with previously published results, we reject the hypothesis that the "non-Bantu" genetic component reported in South-Eastern Africa (Mozambique) reflects extensive gene flow between incoming agriculturalist and resident hunter-gatherer communities. We alternatively suggest that this novel component is the result of demographic dynamics associated with the Bantu dispersal.

Description: Dietary shifts can drive molecular evolution in mammals and a major transition in human history, the agricultural revolution, favored carbohydrate consumption. We investigated the evolutionary history of nine genes encoding brush-border proteins involved in carbohydrate digestion/absorption. Results indicated widespread adaptive evolution in mammals, with several branches experiencing episodic selection, particularly strong in bats. Many positively selected sites map to functional protein regions (e.g., within glucosidase catalytic crevices), with parallel evolution at SI (sucrase-isomaltase) and MGAM (maltase-glucoamylase). In human populations, five genes were targeted by positive selection acting on noncoding variants within regulatory elements. Analysis of ancient DNA samples indicated that most derived alleles were already present in the Paleolithic. Positively selected variants at SLC2A5 (fructose transporter) were an exception and possibly spread following the domestication of specific fruit crops. We conclude that agriculture determined no major selective event at carbohydrate metabolism genes in humans, with implications for susceptibility to metabolic disorders.

Description: Ferns are one of the few remaining major clades of land plants for which a complete genome sequence is lacking. Knowledge of genome space in ferns will enable broad-scale comparative analyses of land plant genes and genomes, provide insights into genome evolution across green plants, and shed light on genetic and genomic features that characterize ferns, such as their high chromosome numbers and large genome sizes. As part of an initial exploration into fern genome space, we used a whole genome shotgun sequencing approach to obtain low-density coverage (~0.4X to 2X) for six fern species from the Polypodiales ( Ceratopteris , Pteridium , Polypodium , Cystopteris ), Cyatheales ( Plagiogyria ), and Gleicheniales ( Dipteris ). We explore these data to characterize the proportion of the nuclear genome represented by repetitive sequences (including DNA transposons, retrotransposons, ribosomal DNA, and simple repeats) and protein-coding genes, and to extract chloroplast and mitochondrial genome sequences. Such initial sweeps of fern genomes can provide information useful for selecting a promising candidate fern species for whole genome sequencing. We also describe variation of genomic traits across our sample and highlight some differences and similarities in repeat structure between ferns and seed plants.

Description: The evolution of heterogametic sex chromosomes is often—but not always—accompanied by the evolution of dosage compensating mechanisms that mitigate the impact of sex-specific gene dosage on levels of gene expression. One emerging view of this process is that such mechanisms may only evolve in male-heterogametic (XY) species but not in female-heterogametic (ZW) species, which will consequently exhibit "incomplete" sex chromosome dosage compensation. However, recent results suggest that at least some Lepidoptera (moths and butterflies) may prove to be an exception to this prediction. Studies in bombycoid moths indicate the presence of a chromosome-wide epigenetic mechanism that effectively balances Z chromosome gene expression between the sexes by reducing Z-linked expression in males. In contrast, strong sex chromosome dosage effects without any reduction in male Z-linked expression were previously reported in a pyralid moth, suggesting a lack of any such dosage compensating mechanism. Here we report an analysis of sex chromosome dosage compensation in Heliconius butterflies, sampling multiple individuals for several different adult tissues (head, abdomen, leg, mouth, and antennae). Methodologically, we introduce a novel application of linear mixed-effects models to assess dosage compensation, offering a unified statistical framework that can estimate effects specific to chromosome, to sex, and their interactions (i.e., a dosage effect). Our results show substantially reduced Z-linked expression relative to autosomes in both sexes, as previously observed in bombycoid moths. This observation is consistent with an increasing body of evidence that some lepidopteran species possess an epigenetic dosage compensating mechanism that reduces Z chromosome expression in males to levels comparable with females. However, this mechanism appears to be imperfect in Heliconius , resulting in a modest dosage effect that produces an average 5–20% increase in male expression relative to females on the Z chromosome, depending on the tissue. Thus our results in Heliconius reflect a mixture of previous patterns reported for Lepidoptera. In Heliconius, a moderate pattern of incomplete dosage compensation persists apparently despite the presence of an epigenetic dosage compensating mechanism. The chromosomal distributions of sex-biased genes show an excess of male-biased and a dearth of female-biased genes on the Z chromosome relative to autosomes, consistent with predictions of sexually antagonistic evolution.

Description: Clonal populations accumulate mutations over time, resulting in different haplotypes. Deep sequencing of such a population in principle provides information to reconstruct these haplotypes and the frequency at which the haplotypes occur. However, this reconstruction is technically not trivial, especially not in clonal systems with a relatively low mutation frequency. The low number of segregating sites in those systems adds ambiguity to the haplotype phasing and thus obviates the reconstruction of genome-wide haplotypes based on sequence overlap information. Therefore, we present EVORhA, a haplotype reconstruction method that complements phasing information in the non-empty read overlap with the frequency estimations of inferred local haplotypes. As was shown with simulated data, as soon as read lengths and/or mutation rates become restrictive for state-of-the-art methods, the use of this additional frequency information allows EVORhA to still reliably reconstruct genome-wide haplotypes. On real data, we show the applicability of the method in reconstructing the population composition of evolved bacterial populations and in decomposing mixed bacterial infections from clinical samples.

Description: Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.

Description: Recent releases of genome three-dimensional (3D) structures have the potential to transform our understanding of genomes. Nonetheless, the storage technology and visualization tools need to evolve to offer to the scientific community fast and convenient access to these data. We introduce simultaneously a database system to store and query 3D genomic data ( 3DBG ), and a 3D genome browser to visualize and explore 3D genome structures ( 3DGB ). We benchmark 3DBG against state-of-the-art systems and demonstrate that it is faster than previous solutions, and importantly gracefully scales with the size of data. We also illustrate the usefulness of our 3D genome Web browser to explore human genome structures. The 3D genome browser is available at http://3dgb.cs.mcgill.ca/ .

Description: Due to their relatively low-cost per sample and broad, gene-centric coverage of CpGs across the human genome, Illumina's 450k arrays are widely used in large scale differential methylation studies. However, by their very nature, large studies are particularly susceptible to the effects of unwanted variation. The effects of unwanted variation have been extensively documented in gene expression array studies and numerous methods have been developed to mitigate these effects. However, there has been much less research focused on the appropriate methodology to use for accounting for unwanted variation in methylation array studies. Here we present a novel 2-stage approach using RUV-inverse in a differential methylation analysis of 450k data and show that it outperforms existing methods.

Description: Telomerase is a reverse transcriptase that maintains telomeres on the ends of chromosomes, allowing rapidly dividing cells to proliferate while avoiding senescence and apoptosis. Understanding telomerase gene expression and splicing at the single cell level could yield insights into the roles of telomerase during normal cell growth as well as cancer development. Here we use droplet-based single cell culture followed by single cell or colony transcript abundance analysis to investigate the relationship between cell growth and transcript abundance of the telomerase genes encoding the RNA component (hTR) and protein component (hTERT) as well as hTERT splicing. Jurkat and K562 cells were examined under normal cell culture conditions and during exposure to curcumin, a natural compound with anti-carcinogenic and telomerase activity-reducing properties. Individual cells predominantly express single hTERT splice variants, with the α+/β– variant exhibiting significant transcript abundance bimodality that is sustained through cell division. Sub-lethal curcumin exposure results in reduced bimodality of all hTERT splice variants and significant upregulation of alpha splicing, suggesting a possible role in cellular stress response. The single cell culture and transcript abundance analysis method presented here provides the tools necessary for multiparameter single cell analysis which will be critical for understanding phenotypes of heterogeneous cell populations, disease cell populations and their drug response.

Description: Mycobacterium avium ( M. a. ) subsp. paratuberculosis (MAP)—the etiologic agent of Johne’s disease—affects cattle, sheep, and other ruminants worldwide. To decipher phenotypic differences among sheep and cattle strains (belonging to MAP-S [Type-I/III], respectively, MAP-C [Type-II]), comparative genome analysis needs data from diverse isolates originating from different geographic regions of the world. This study presents the so far best assembled genome of a MAP-S-strain: Sheep isolate JIII-386 from Germany. One newly sequenced cattle isolate (JII-1961, Germany), four published MAP strains of MAP-C and MAP-S from the United States and Australia, and M. a. subsp. hominissuis (MAH) strain 104 were used for assembly improvement and comparisons. All genomes were annotated by BacProt and results compared with NCBI (National Center for Biotechnology Information) annotation. Corresponding protein-coding sequences (CDSs) were detected, but also CDSs that were exclusively determined by either NCBI or BacProt. A new Shine–Dalgarno sequence motif (5'-AGCTGG-3') was extracted. Novel CDSs including PE-PGRS family protein genes and about 80 noncoding RNAs exhibiting high sequence conservation are presented. Previously found genetic differences between MAP-types are partially revised. Four of ten assumed MAP-S-specific large sequence polymorphism regions (LSP S s) are still present in MAP-C strains; new LSP S s were identified. Independently of the regional origin of the strains, the number of individual CDSs and single nucleotide variants confirms the strong similarity of MAP-C strains and shows higher diversity among MAP-S strains. This study gives ambiguous results regarding the hypothesis that MAP-S is the evolutionary intermediate between MAH and MAP-C, but it clearly shows a higher similarity of MAP to MAH than to Mycobacterium intracellulare .

Description: Eukaryotic organelles depend on nuclear genes to perpetuate their biochemical integrity. This is true for mitochondria in all eukaryotes and plastids in plants and algae. Then how do kleptoplasts, plastids that are sequestered by some sacoglossan sea slugs, survive in the animals’ digestive gland cells in the absence of the algal nucleus encoding the vast majority of organellar proteins? For almost two decades, lateral gene transfer (LGT) from algae to slugs appeared to offer a solution, but RNA-seq analysis, later supported by genome sequencing of slug DNA, failed to find any evidence for such LGT events. Yet, isolated reports continue to be published and are readily discussed by the popular press and social media, making the data on LGT and its support for kleptoplast longevity appear controversial. However, when we take a sober look at the methods used, we realize that caution is warranted in how the results are interpreted. There is no evidence that the evolution of kleptoplasty in sea slugs involves LGT events. Based on what we know about photosystem maintenance in embryophyte plastids, we assume kleptoplasts depend on nuclear genes. However, studies have shown that some isolated algal plastids are, by nature, more robust than those of land plants. The evolution of kleptoplasty in green sea slugs involves many promising and unexplored phenomena, but there is no evidence that any of these require the expression of slug genes of algal origin.

Description: A number of sap-sucking insects harbor endosymbionts, which are thought to play an important role in the development of their hosts. One of the most important rice pests, the brown planthopper (BPH), Nilaparvata lugens (Stål), harbors an obligatory yeast-like symbiont (YLS) that cannot be cultured in vitro. Genomic information on this YLS would be useful to better understand its evolution. In this study, we performed genome sequencing of the YLS using both 454 and Illumina approaches, generating a draft genome that shows a slightly smaller genome size and relatively higher GC content than most ascomycete fungi. A phylogenomic analysis of the YLS supported its close relationship with insect pathogens. We analyzed YLS-specific genes and the categories of genes that are likely to have changed in the YLS during its evolution. The loss of mating type locus demonstrated in the YLS sheds light on the evolution of eukaryotic symbionts. This information about the YLS genome provides a helpful guide for further understanding endosymbiotic associations in hemiptera and the symbiotic replacement of ancient bacteria with a multifunctional YLS seems to have been a successful change.

Description: The RNA-directed DNA methylation (RdDM) pathway can be divided into three phases: 1) small interfering RNA biogenesis, 2) de novo methylation, and 3) chromatin modification. To determine the degree of conservation of this pathway we searched for key genes among land plants. We used OrthoMCL and the OrthoMCL Viridiplantae database to analyze proteomes of species in bryophytes, lycophytes, monilophytes, gymnosperms, and angiosperms. We also analyzed small RNA size categories and, in two gymnosperms, cytosine methylation in ribosomal DNA. Six proteins were restricted to angiosperms, these being NRPD4/NRPE4, RDM1, DMS3 (defective in meristem silencing 3), SHH1 (SAWADEE homeodomain homolog 1), KTF1, and SUVR2, although we failed to find the latter three proteins in Fritillaria persica , a species with a giant genome. Small RNAs of 24 nt in length were abundant only in angiosperms. Phylogenetic analyses of Dicer-like (DCL) proteins showed that DCL2 was restricted to seed plants, although it was absent in Gnetum gnemon and Welwitschia mirabilis . The data suggest that phases (1) and (2) of the RdDM pathway, described for model angiosperms, evolved with angiosperms. The absence of some features of RdDM in F. persica may be associated with its large genome. Phase (3) is probably the most conserved part of the pathway across land plants. DCL2, involved in virus defense and interaction with the canonical RdDM pathway to facilitate methylation of CHH, is absent outside seed plants. Its absence in G. gnemon , and W. mirabilis coupled with distinctive patterns of CHH methylation, suggest a secondary loss of DCL2 following the divergence of Gnetales.

Description: Here, we present a simple, modular and efficient strategy that allows the 3'-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3'-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation.

Description: Despite the increasing knowledge about DNA methylation, the understanding of human epigenome evolution is in its infancy. Using whole genome bisulfite sequencing we identified hundreds of differentially methylated regions (DMRs) in humans compared to non-human primates and estimated that ~25% of these regions were detectable throughout several human tissues. Human DMRs were enriched for specific histone modifications and the majority were located distal to transcription start sites, highlighting the importance of regions outside the direct regulatory context. We also found a significant excess of endogenous retrovirus elements in human-specific hypomethylated. We reported for the first time a close interplay between inter-species genetic and epigenetic variation in regions of incomplete lineage sorting, transcription factor binding sites and human differentially hypermethylated regions. Specifically, we observed an excess of human-specific substitutions in transcription factor binding sites located within human DMRs, suggesting that alteration of regulatory motifs underlies some human-specific methylation patterns. We also found that the acquisition of DNA hypermethylation in the human lineage is frequently coupled with a rapid evolution at nucleotide level in the neighborhood of these CpG sites. Taken together, our results reveal new insights into the mechanistic basis of human-specific DNA methylation patterns and the interpretation of inter-species non-coding variation.

Description: The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum . We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family.

Description: MicroRNAs are a class of small regulatory RNAs that are generated from primary miRNA (pri-miRNA) transcripts with a stem-loop structure. Accuracy of the processing of pri-miRNA into mature miRNA in plants can be enhanced by SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1). HYL1 activity is regulated by the FIERY2 (FRY2)/RNA polymerase II C-terminal domain phosphatase-like 1 (CPL1). Here, we discover that HIGH OSMOTIC STRESS GENE EXPRESSION 5 (HOS5) and two serine/arginine-rich splicing factors RS40 and RS41, previously shown to be involved in pre-mRNA splicing, affect the biogenesis of a subset of miRNA. These proteins are required for correct miRNA strand selection and the maintenance of miRNA levels. FRY2 dephosphorylates HOS5 whose phosphorylation status affects its subnuclear localization. HOS5 and the RS proteins bind both intronless and intron-containing pri-miRNAs. Importantly, all of these splicing-related factors directly interact with both HYL1 and SE in nuclear splicing speckles. Our results indicate that these splicing factors are directly involved in the biogenesis of a group of miRNA.

Description: AlgR is a key transcriptional regulator required for the expression of multiple virulence factors, including type IV pili and alginate in Pseudomonas aeruginosa . However, the regulon and molecular regulatory mechanism of AlgR have yet to be fully elucidated. Here, among 157 loci that were identified by a ChIP-seq assay, we characterized a gene, mucR , which encodes an enzyme that synthesizes the intracellular second messenger cyclic diguanylate (c-di-GMP). A algR strain produced lesser biofilm than did the wild-type strain, which is consistent with a phenotype controlled by c-di-GMP. AlgR positively regulates mucR via direct binding to its promoter. A algR mucR double mutant produced lesser biofilm than did the single algR mutant, demonstrating that c-di-GMP is a positive regulator of biofilm formation. AlgR controls the levels of c-di-GMP synthesis via direct regulation of mucR . In addition, the cognate sensor of AlgR, FimS/AlgZ, also plays an important role in P. aeruginosa virulence. Taken together, this study provides new insights into the AlgR regulon and reveals the involvement of c-di-GMP in the mechanism underlying AlgR regulation.

Description: Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II (Pol II), especially Ser2 and Ser5 residues, plays important roles in transcription and mRNA processing, including 5' end capping, splicing and 3' end processing. These phosphorylation events stimulate mRNA processing, however, it is not clear whether splicing activity affects the phosphorylation status of Pol II. In this study, we found that splicing inhibition by potent splicing inhibitors spliceostatin A (SSA) and pladienolide B or by antisense oligos against snRNAs decreased phospho-Ser2 level, but had little or no effects on phospho-Ser5 level. In contrast, transcription and translation inhibitors did not decrease phospho-Ser2 level, therefore inhibition of not all the gene expression processes cause the decrease of phospho-Ser2. SSA treatment caused early dissociation of Pol II and decrease in phospho-Ser2 level of chromatin-bound Pol II, suggesting that splicing inhibition causes downregulation of phospho-Ser2 through at least these two mechanisms.

Description: Facioscapulohumeral dystrophy (FSHD) is an epi/genetic satellite disease associated with at least two satellite sequences in 4q35: (i) D4Z4 macrosatellite and (ii) β-satellite repeats (BSR), a prevalent part of the 4qA allele. Most of the recent FSHD studies have been focused on a DUX4 transcript inside D4Z4 and its tandem contraction in FSHD patients. However, the D4Z4-contraction alone is not pathological, which would also require the 4qA allele. Since little is known about BSR, we investigated the 4qA BSR functional role in the transcriptional control of the FSHD region 4q35. We have shown that an individual BSR possesses enhancer activity leading to activation of the Adenine Nucleotide Translocator 1 gene ( ANT1 ), a major FSHD candidate gene. We have identified ZNF555, a previously uncharacterized protein, as a putative transcriptional factor highly expressed in human primary myoblasts that interacts with the BSR enhancer site and impacts the ANT1 promoter activity in FSHD myoblasts. The discovery of the functional role of the 4qA allele and ZNF555 in the transcriptional control of ANT1 advances our understanding of FSHD pathogenesis and provides potential therapeutic targets.

Description: The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8–2'-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G 1 -, G 2 - or G 3 -positions of the Nar I site (5'-G 1 G 2 CG 3 CC-3') were replicated in HEK293T cells. Fifty percent of the progeny from the G 3 construct were mutants, largely G-〉T, compared to 18% and 24% from the G 1 and G 2 constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38–67% upon siRNA knockdown of pol , whereas it was increased by 10–24% in pol knockdown cells. When pol and pol were simultaneously knocked down, MF of the G 1 and G 3 constructs was reduced from 18% and 50%, respectively, to 〈3%, whereas it was reduced from 24% to 〈1% in the G 2 construct. In vitro TLS using yeast pol showed that it can extend G 3 *:A pair more efficiently than G 3 *:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol and pol cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol is involved primarily in its error-free bypass.

Description: The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

Description: To gain a wider view of the pathways that regulate mitochondrial function, we combined the effect of heat stress on respiratory capacity with the discovery potential of a genome-wide screen in Saccharomyces cerevisiae . We identified 105 new genes whose deletion impairs respiratory growth at 37°C by interfering with processes such as transcriptional regulation, ubiquitination and cytosolic tRNA wobble uridine modification via 5-methoxycarbonylmethyl-2-thiouridine formation. The latter process, specifically required for efficient decoding of AA-ending codons under stress conditions, was covered by multiple genes belonging to the Elongator (e.g. ELP3 ) and urmylation (e.g., NCS6 ) pathways. ELP3 or NCS6 deletants had impaired mitochondrial protein synthesis. Their respiratory deficiency was selectively rescued by overexpression of tRNA Lys UUU as well by overexpression of genes ( BCK1 and HFM1 ) with a strong bias for the AAA codon read by this tRNA. These data extend the mitochondrial regulome, demonstrate that heat stress can impair respiration by disturbing cytoplasmic translation of proteins critically involved in mitochondrial function and document, for the first time, the involvement in such process of the Elongator and urmylation pathways. Given the conservation of these pathways, the present findings may pave the way to a better understanding of the human mitochondrial regulome in health and disease.

Description: Recent studies strongly suggest that in bacterial cells the order of genes along the chromosomal origin-to-terminus axis is determinative for regulation of the growth phase-dependent gene expression. The prediction from this observation is that positional displacement of pleiotropic genes will affect the genetic regulation and hence, the cellular phenotype. To test this prediction we inserted the origin-proximal dusB-fis operon encoding the global regulator FIS in the vicinity of replication terminus on both arms of the Escherichia coli chromosome. We found that the lower fis gene dosage in the strains with terminus-proximal dusB-fis operons was compensated by increased fis expression such that the intracellular concentration of FIS was homeostatically adjusted. Nevertheless, despite unchanged FIS levels the positional displacement of dusB-fis impaired the competitive growth fitness of cells and altered the state of the overarching network regulating DNA topology, as well as the cellular response to environmental stress, hazardous substances and antibiotics. Our finding that the chromosomal repositioning of a regulatory gene can determine the cellular phenotype unveils an important yet unexplored facet of the genetic control mechanisms and paves the way for novel approaches to manipulate bacterial physiology.

Description: In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli , short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro . Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.

Description: DNA damage, arising from endogenous metabolism or exposure to environmental agents, may perturb the transmission of genetic information by blocking DNA replication and/or inducing mutations, which contribute to the development of cancer and likely other human diseases. Hydroxyl radical attack on the C1', C3' and C4' of 2-deoxyribose can give rise to epimeric 2-deoxyribose lesions, for which the in vivo occurrence and biological consequences remain largely unexplored. Through independent chemical syntheses of all three epimeric lesions of 2'-deoxyguanosine (dG) and liquid chromatography-tandem mass spectrometry analysis, we demonstrated unambiguously the presence of substantial levels of the α-anomer of dG (α-dG) in calf thymus DNA and in DNA isolated from mouse pancreatic tissues. We further assessed quantitatively the impact of all four α-dN lesions on DNA replication in Escherichia coli by employing a shuttle-vector method. We found that, without SOS induction, all α-dN lesions except α-dA strongly blocked DNA replication and, while replication across α-dA was error-free, replicative bypass of α-dC and α-dG yielded mainly C-〉A and G-〉A mutations. In addition, SOS induction could lead to markedly elevated bypass efficiencies for the four α-dN lesions, abolished the G-〉A mutation for α-dG, pronouncedly reduced the C-〉A mutation for α-dC and triggered T-〉A mutation for α-dT. The preferential misincorporation of dTMP opposite the α-dNs could be attributed to the unique base-pairing properties of the nucleobases elicited by the inversion of the configuration of the N -glycosidic linkage. Our results also revealed that Pol V played a major role in bypassing α-dC, α-dG and α-dT in vivo . The abundance of α-dG in mammalian tissue and the impact of the α-dNs on DNA replication demonstrate for the first time the biological significance of this family of DNA lesions.

Description: We have examined the three-dimensional organization of the yeast genome during quiescence by a chromosome capture technique as a means of understanding how genome organization changes during development. For exponentially growing cells we observe high levels of inter-centromeric interaction but otherwise a predominance of intrachromosomal interactions over interchromosomal interactions, consistent with aggregation of centromeres at the spindle pole body and compartmentalization of individual chromosomes within the nucleoplasm. Three major changes occur in the organization of the quiescent cell genome. First, intrachromosomal associations increase at longer distances in quiescence as compared to growing cells. This suggests that chromosomes undergo condensation in quiescence, which we confirmed by microscopy by measurement of the intrachromosomal distances between two sites on one chromosome. This compaction in quiescence requires the condensin complex. Second, inter-centromeric interactions decrease, consistent with prior data indicating that centromeres disperse along an array of microtubules during quiescence. Third, inter-telomeric interactions significantly increase in quiescence, an observation also confirmed by direct measurement. Thus, survival during quiescence is associated with substantial topological reorganization of the genome.

Description: Cross-talk between competitive endogenous RNAs (ceRNAs) through shared miRNAs represents a novel layer of gene regulation that plays important roles in the physiology and development of cancers. However, a global view of their system-level properties across various types of cancers is still unknown. Here, we constructed the mRNA related ceRNA–ceRNA interaction landscape across 20 cancer types by systematically analyzing molecular profiles of 5203 tumors and miRNA regulations. Our study highlights the conserved features shared by pan-cancer and higher similarity within similar origin cell type. Moreover, a core ceRNA network was identified. Function analysis identified a common theme of cancer hallmarks, however they exhibit phenotype-specific connectivity patterns. Besides, we found a marked rewiring in the ceRNA program between various cancers, and further revealed conserved and rewired network ceRNA hubs in each cancer, which were tensely competitive interactions to constitute conserved and cancer-specific modules. By providing mechanistic linkage between known cancer miRNAs, their mediated ceRNA–ceRNA interactions, and the associations with known cancer hallmarks, the inferred cancer ceRNA–ceRNA interaction landscape will serve as a powerful public resource for further biological discoveries of tumorigenesis.

Description: Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB ( Hp DnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of Hp DnaB protein during primosome formation. The crystal structure of the Hp DnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that Hp DnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of Hp DnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.

Description: Microcalorimetric studies of DNA duplexes and their component single strands showed that association enthalpies of unfolded complementary strands into completely folded duplexes increase linearly with temperature and do not depend on salt concentration, i.e. duplex formation results in a constant heat capacity decrement, identical for CG and AT pairs. Although duplex thermostability increases with CG content, the enthalpic and entropic contributions of an AT pair to duplex formation exceed that of a CG pair when compared at the same temperature. The reduced contribution of AT pairs to duplex stabilization comes not from their lower enthalpy, as previously supposed, but from their larger entropy contribution. This larger enthalpy and particularly the greater entropy results from water fixed by the AT pair in the minor groove. As the increased entropy of an AT pair exceeds that of melting ice, the water molecule fixed by this pair must affect those of its neighbors. Water in the minor groove is, thus, orchestrated by the arrangement of AT groups, i.e. is context dependent. In contrast, water hydrating exposed nonpolar surfaces of bases is responsible for the heat capacity increment on dissociation and, therefore, for the temperature dependence of all thermodynamic characteristics of the double helix.

Description: Small RNAs (sRNAs) are important regulators of gene expression during bacterial stress and pathogenesis. sRNAs act by forming duplexes with mRNAs to alter their translation and degradation. In some bacteria, duplex formation is mediated by the Hfq protein, which can bind the sRNA and mRNA in each pair in a random order. Here we investigate the consequences of this random-order binding and experimentally demonstrate that it can counterintuitively cause high Hfq concentrations to suppress rather than promote sRNA activity in Escherichia coli . As a result, maximum sRNA activity occurs when the Hfq concentration is neither too low nor too high relative to the sRNA and mRNA concentrations (‘Hfq set-point’). We further show with models and experiments that random-order binding combined with the formation of a dead-end mRNA–Hfq complex causes high concentrations of an mRNA to inhibit its own duplex formation by sequestering Hfq. In such cases, maximum sRNA activity requires an optimal mRNA concentration (‘mRNA set-point’) as well as an optimal Hfq concentration. The Hfq and mRNA set-points generate novel regulatory properties that can be harnessed by native and synthetic gene circuits to provide greater control over sRNA activity, generate non-monotonic responses and enhance the robustness of expression.

Description: A prolonged expansion of GGGGCC repeat within non-coding region of C9orf72 gene has been identified as the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are devastating neurodegenerative disorders. Formation of unusual secondary structures within expanded GGGGCC repeat, including DNA and RNA G-quadruplexes and R-loops was proposed to drive ALS and FTD pathogenesis. Initial NMR investigation on DNA oligonucleotides with four repeat units as the shortest model with the ability to form an unimolecular G-quadruplex indicated their folding into multiple G-quadruplex structures in the presence of K + ions. Single dG to 8Br-dG substitution at position 21 in oligonucleotide d[(G 4 C 2 ) 3 G 4 ] and careful optimization of folding conditions enabled formation of mostly a single G-quadruplex species, which enabled determination of a high-resolution structure with NMR. G-quadruplex structure adopted by d[(G 4 C 2 ) 3 GG Br GG] is composed of four G-quartets, which are connected by three edgewise C-C loops. All four strands adopt antiparallel orientation to one another and have alternating syn-anti progression of glycosidic conformation of guanine residues. One of the cytosines in every loop is stacked upon the G-quartet contributing to a very compact and stable structure.

Description: Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania , resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)—the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3 , as a prospective therapeutic candidate for the treatment of VL.

Description: Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by the silencing of the FMR1 gene encoding an RNA-binding protein (FMRP) mainly involved in translational control. We characterized the interaction between FMRP and the mRNA of GRK4 , a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase super-family, both in vitro and in vivo . While the mRNA level of GRK4 is unchanged in the absence or in the presence of FMRP in different regions of the brain, GRK4 protein level is increased in Fmr1 -null cerebellum, suggesting that FMRP negatively modulates the expression of GRK4 at the translational level in this brain region. The C-terminal region of FMRP interacts with a domain of GRK4 mRNA, that we called G4RIF, that is folded in four stem loops. The SL1 stem loop of G4RIF is protected by FMRP and is part of the S1/S2 sub-domain that directs translation repression of a reporter mRNA by FMRP. These data confirm the role of the G4RIF/FMRP complex in translational regulation. Considering the role of GRK4 in GABAB receptors desensitization, our results suggest that an increased GRK4 levels in FXS might contribute to cerebellum-dependent phenotypes through a deregulated desensitization of GABAB receptors.

Description: Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe , amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ~3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures.

Description: Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5' ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3' ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5' passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.

Description: Comparisons of draft genome sequences of three geographically distinct isolates of Fusarium fujikuroi with two recently published genome sequences from the same species suggest diverse profiles of secondary metabolite production within F. fujikuroi . Species- and lineage-specific genes, many of which appear to exhibit expression profiles that are consistent with roles in host–pathogen interactions and adaptation to environmental changes, are concentrated in subtelomeric regions. These genomic compartments also exhibit distinct gene densities and compositional characteristics with respect to other genomic partitions, and likely play a role in the generation of molecular diversity. Our data provide additional evidence that gene duplication, divergence, and differential loss play important roles in F. fujikuroi genome evolution and suggest that hundreds of lineage-specific genes might have been acquired through horizontal gene transfer.

Description: Passerines are the largest avian order, and the 6,000 species comprise more than half of all extant bird species. This successful radiation probably had its origin in the Australasian region, but dating this origin has been difficult due to a scarce fossil record and poor biogeographic assumptions. Many of New Zealand’s endemic passerines fall within the deeper branches of the passerine radiation, and a well resolved phylogeny for the modern New Zealand element in the deeper branches of the oscine lineage will help us understand both oscine and passerine biogeography. To this end we present complete mitochondrial genomes representing all families of New Zealand passerines in a phylogenetic framework of over 100 passerine species. Dating analyses of this robust phylogeny suggest Passeriformes originated in the early Paleocene, with the major lineages of oscines "escaping" from Australasia about 30 Ma, and radiating throughout the world during the Oligocene. This independently derived conclusion is consistent with the passerine fossil record.

Description: Triplophysa fishes are the primary component of the fish fauna on the Tibetan Plateau and are well adapted to the high-altitude environment. Despite the importance of Triplophysa fishes on the plateau, the genetic mechanisms of the adaptations of these fishes to this high-altitude environment remain poorly understood. In this study, we generated the transcriptome sequences for three Triplophysa fishes, that is, Triplophysa siluroides , Triplophysa scleroptera , and Triplophysa dalaica , and used these and the previously available transcriptome and genome sequences from fishes living at low altitudes to identify potential genetic mechanisms for the high-altitude adaptations in Triplophysa fishes. An analysis of 2,269 orthologous genes among cave fish ( Astyanax mexicanus ), zebrafish ( Danio rerio ), large-scale loach ( Paramisgurnus dabryanus ), and Triplophysa fishes revealed that each of the terminal branches of the Triplophysa fishes had a significantly higher ratio of nonsynonymous to synonymous substitutions than that of the branches of the fishes from low altitudes, which provided consistent evidence for genome-wide rapid evolution in the Triplophysa genus. Many of the GO (Gene Ontology) categories associated with energy metabolism and hypoxia response exhibited accelerated evolution in the Triplophysa fishes compared with the large-scale loach. The genes that exhibited signs of positive selection and rapid evolution in the Triplophysa fishes were also significantly enriched in energy metabolism and hypoxia response categories. Our analysis identified widespread Triplophysa -specific nonsynonymous mutations in the fast evolving genes and positively selected genes. Moreover, we detected significant evidence of positive selection in the HIF (hypoxia-inducible factor)-1A and HIF-2B genes in Triplophysa fishes and found that the Triplophysa -specific nonsynonymous mutations in the HIF-1A and HIF-2B genes were associated with functional changes. Overall, our study provides new insights into the adaptations and evolution of fishes in the high-altitude environment of the Tibetan Plateau and complements previous findings on the adaptations of mammals and birds to high altitudes.

Description: Sponges harbor a complex consortium of microbial communities living in symbiotic relationship benefiting each other through the integration of metabolites. The mechanisms influencing a successful microbial association with a sponge partner are yet to be fully understood. Here, we sequenced the genome of Pseudovibrio sp. POLY-S9 strain isolated from the intertidal marine sponge Polymastia penicillus sampled from the Atlantic coast of Portugal to identify the genomic features favoring the symbiotic relationship. The draft genome revealed an exceptionally large genome size of 6.6 Mbp compared with the previously reported genomes of the genus Pseudovibrio isolated from a coral and a sponge larva. Our genomic study detected the presence of several biosynthetic gene clusters—polyketide synthase, nonribosomal peptide synthetase and siderophore—affirming the potential ability of the genus Pseudovibrio to produce a wide variety of metabolic compounds. Moreover, we identified a repertoire of genes encoding adaptive symbioses factors (eukaryotic-like proteins), such as the ankyrin repeats, tetratrico peptide repeats, and Sel1 repeats that improve the attachment to the eukaryotic hosts and the avoidance of the host’s immune response . The genome also harbored a large number of mobile elements (~5%) and gene transfer agents, which explains the massive genome expansion and suggests a possible mechanism of horizontal gene transfer. In conclusion, the genome of POLY-S9 exhibited an increase in size, number of mobile DNA, multiple metabolite gene clusters, and secretion systems, likely to influence the genome diversification and the evolvability.

Description: Gene regulatory networks (GRN) are central to developmental processes. They are composed of transcription factors and signaling molecules orchestrating gene expression modules that tightly regulate the development of organisms. The neural crest (NC) is a multipotent cell population that is considered a key innovation of vertebrates. Its derivatives contribute to shaping the astounding morphological diversity of jaws, teeth, head skeleton, or pigmentation. Here, we study the molecular evolution of the NC GRN by analyzing patterns of molecular divergence for a total of 36 genes in 16 species of bony fishes. Analyses of nonsynonymous to synonymous substitution rate ratios (d N /d S ) support patterns of variable selective pressures among genes deployed at different stages of NC development, consistent with the developmental hourglass model. Model-based clustering techniques of sequence features support the notion of extreme conservation of NC-genes across the entire network. Our data show that most genes are under strong purifying selection that is maintained throughout ray-finned fish evolution. Late NC development genes reveal a pattern of increased constraints in more recent lineages. Additionally, seven of the NC-genes showed signs of relaxation of purifying selection in the famously species-rich lineage of cichlid fishes. This suggests that NC genes might have played a role in the adaptive radiation of cichlids by granting flexibility in the development of NC-derived traits—suggesting an important role for NC network architecture during the diversification in vertebrates.

Description: Cymbomonas tetramitiformis —a marine prasinophyte—is one of only a few green algae that still retain an ancestral particulate-feeding mechanism while harvesting energy through photosynthesis. The genome of the alga is estimated to be 850 Mb–1.2 Gb in size—the bulk of which is filled with repetitive sequences—and is annotated with 37,366 protein-coding gene models. A number of unusual metabolic pathways (for the Chloroplastida) are predicted for C. tetramitiformis , including pathways for Lipid-A and peptidoglycan metabolism. Comparative analyses of the predicted peptides of C. tetramitiformis to sets of other eukaryotes revealed that nonphagocytes are depleted in a number of genes, a proportion of which have known function in feeding. In addition, our analysis suggests that obligatory phagotrophy is associated with the loss of genes that function in biosynthesis of small molecules (e.g., amino acids). Further, C. tetramitiformis and at least one other phago-mixotrophic alga are thus unique, compared with obligatory heterotrophs and nonphagocytes, in that both feeding and small molecule synthesis-related genes are retained in their genomes. These results suggest that early, ancestral host eukaryotes that gave rise to phototrophs had the capacity to assimilate building block molecules from inorganic substances (i.e., prototrophy). The loss of biosynthesis genes, thus, may at least partially explain the apparent lack of instances of permanent incorporation of photosynthetic endosymbionts in later-divergent, auxotrophic eukaryotic lineages, such as metazoans and ciliates.

Description: Chlamydiaceae are obligate intracellular bacteria that cause a diversity of severe infections among humans and livestock on a global scale. Identification of new species since 1989 and emergence of zoonotic infections, including abortion in women, underscore the need for genome sequencing of multiple strains of each species to advance our knowledge of evolutionary dynamics across Chlamydiaceae . Here, we genome sequenced isolates from avian, lower mammalian and human hosts. Based on core gene phylogeny, five isolates previously classified as Chlamydia abortus were identified as members of Chlamydia psittaci and Chlamydia pecorum. Chlamydia abortus is the most recently emerged species and is a highly monomorphic group that lacks the conserved virulence-associated plasmid. Low-level recombination and evidence for adaptation to the placenta echo evolutionary processes seen in recently emerged, highly virulent niche-restricted pathogens, such as Bacillus anthracis . In contrast, gene flow occurred within C. psittaci and other Chlamydiaceae species. The C. psittaci strain RTH, isolated from a red-tailed hawk ( Buteo jamaicensis ), is an outlying strain with admixture of C. abortus , C. psittaci , and its own population markers. An average nucleotide identity of less than 94% compared with other Chlamydiaceae species suggests that RTH belongs to a new species intermediary between C. psittaci and C. abortus . Hawks, as scavengers and predators, have extensive opportunities to acquire multiple species in their intestinal tract. This could facilitate transformation and homologous recombination with the potential for new species emergence. Our findings indicate that incubator hosts such as birds-of-prey likely promote Chlamydiaceae evolution resulting in novel pathogenic lineages.

Description: Data on biological mechanisms of aging are mostly obtained from cross-sectional study designs. An inherent disadvantage of this design is that inter-individual differences can mask small but biologically significant age-dependent changes. A serially sampled design (same individual at different time points) would overcome this problem but is often limited by the relatively small numbers of available paired samples and the statistics being used. To overcome these limitations, we have developed a new vector-based approach, termed three-component analysis, which incorporates temporal distance, signal intensity and variance into one single score for gene ranking and is combined with gene set enrichment analysis. We tested our method on a unique age-based sample set of human skin fibroblasts and combined genome-wide transcription, DNA methylation and histone methylation (H3K4me3 and H3K27me3) data. Importantly, our method can now for the first time demonstrate a clear age-dependent decrease in expression of genes coding for proteins involved in translation and ribosome function. Using analogies with data from lower organisms, we propose a model where age-dependent down-regulation of protein translation-related components contributes to extend human lifespan.

Description: With the development of High-Throughput Sequencing (HTS) thousands of human genomes have now been sequenced. Whenever different studies analyze the same genome they usually agree on the amount of single-nucleotide polymorphisms, but differ dramatically on the number of insertion and deletion variants (indels). Furthermore, there is evidence that indels are often severely under-reported. In this manuscript we derive the total number of indel variants in a human genome by combining data from different sequencing technologies, while assessing the indel detection accuracy. Our estimate of approximately 1 million indels in a Yoruban genome is much higher than the results reported in several recent HTS studies. We identify two key sources of difficulties in indel detection: the insufficient coverage, read length or alignment quality; and the presence of repeats, including short interspersed elements and homopolymers/dimers. We quantify the effect of these factors on indel detection. The quality of sequencing data plays a major role in improving indel detection by HTS methods. However, many indels exist in long homopolymers and repeats, where their detection is severely impeded. The true number of indel events is likely even higher than our current estimates, and new techniques and technologies will be required to detect them.

Description: Polyamines are ubiquitous cations that are involved in regulating fundamental cellular processes such as cell growth and proliferation; hence, their intracellular concentration is tightly regulated. Antizyme and antizyme inhibitor have a central role in maintaining cellular polyamine levels. Antizyme is unique in that it is expressed via a novel programmed ribosomal frameshifting mechanism. Conventional computational tools are unable to predict a programmed frameshift, resulting in misannotation of antizyme transcripts and proteins on transcript and genomic sequences. Correct annotation of a programmed frameshifting event requires manual evaluation. Our goal was to provide an accurately curated and annotated Reference Sequence (RefSeq) data set of antizyme transcript and protein records across a broad taxonomic scope that would serve as standards for accurate representation of these gene products. As antizyme and antizyme inhibitor proteins are functionally connected, we also curated antizyme inhibitor genes to more fully represent the elegant biology of polyamine regulation. Manual review of genes for three members of the antizyme family and two members of the antizyme inhibitor family in 91 vertebrate organisms resulted in a total of 461 curated RefSeq records.

Description: Due to the helical structure of DNA the process of DNA replication is topologically complex. Freshly replicated DNA molecules are catenated with each other and are frequently knotted. For proper functioning of DNA it is necessary to remove all of these entanglements. This is done by DNA topoisomerases that pass DNA segments through each other. However, it has been a riddle how DNA topoisomerases select the sites of their action. In highly crowded DNA in living cells random passages between contacting segments would only increase the extent of entanglement. Using molecular dynamics simulations we observed that in actively supercoiled DNA molecules the entanglements resulting from DNA knotting or catenation spontaneously approach sites of nicks and gaps in the DNA. Type I topoisomerases, that preferentially act at sites of nick and gaps, are thus naturally provided with DNA–DNA juxtapositions where a passage results in an error-free DNA unknotting or DNA decatenation.

Description: TrmJ proteins from the SPOUT methyltransferase superfamily are tRNA Xm32 modification enzymes that occur in bacteria and archaea. Unlike archaeal TrmJ, bacterial TrmJ require full-length tRNA molecules as substrates. It remains unknown how bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2'- O modification at ribose 32. Herein, we demonstrate that all six Escherichia coli ( Ec ) tRNAs with 2'- O -methylated nucleosides at position 32 are substrates of Ec TrmJ, and we show that the elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. Our crystallographic study reveals that full-length Ec TrmJ forms an unusual dimer in the asymmetric unit, with both the catalytic SPOUT domain and C-terminal extension forming separate dimeric associations. Based on these findings, we used electrophoretic mobility shift assay, isothermal titration calorimetry and enzymatic methods to identify amino acids within Ec TrmJ that are involved in tRNA binding. We found that tRNA recognition by Ec TrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and that this process is regulated by the flexible hinge region that connects these two domains.

Description: Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity ( K d ~12-30 nM) selective competitive inhibitors (IC 50  ~20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors.

Description: To define the functions of NCOR1 we developed an integrative analysis that combined ENCODE and NCI-60 data, followed by in vitro validation. NCOR1 and H3K9me3 ChIP-Seq, FAIRE-seq and DNA CpG methylation interactions were related to gene expression using bootstrapping approaches. Most NCOR1 combinations (24/44) were associated with significantly elevated level expression of protein coding genes and only very few combinations related to gene repression. DAVID's biological process annotation revealed that elevated gene expression was uniquely associated with acetylation and ETS binding. A matrix of gene and drug interactions built on NCI-60 data identified that Imatinib significantly targeted the NCOR1 governed transcriptome. Stable knockdown of NCOR1 in K562 cells slowed growth and significantly repressed genes associated with NCOR1 cistrome, again, with the GO terms acetylation and ETS binding, and significantly dampened sensitivity to Imatinib-induced erythroid differentiation. Mining public microarray data revealed that NCOR1-targeted genes were significantly enriched in Imatinib response gene signatures in cell lines and chronic myelogenous leukemia (CML) patients. These approaches integrated cistrome, transcriptome and drug sensitivity relationships to reveal that NCOR1 function is surprisingly most associated with elevated gene expression, and that these targets, both in CML cell lines and patients, associate with sensitivity to Imatinib.

Description: On average, mutations are deleterious to proteins. Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity. Over the years, a panel of T7 RNA polymerases have been designed or evolved to accept nucleotides with modified ribose moieties. These modified RNAs have proven useful, especially in vivo , but the transcriptional yields tend to be quite low. Here we show that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range. The resulting polymerase mutants can be used to generate 2' -O- methyl modified RNA with yields much higher than enzymes currently employed.

Description: Interactions of RNA-binding proteins (RBPs) with their target transcripts are essential for regulating gene expression at the posttranscriptional level including mRNA export/localization, stability, and translation. ZBP1 and HuD are RBPs that play pivotal roles in mRNA transport and local translational control in neuronal processes. While HuD possesses three RNA recognition motifs (RRMs), ZBP1 contains two RRMs and four K homology (KH) domains that either increase target specificity or provide a multi-target binding capability. Here we used isolated cis -element sequences of the target mRNA to examine directly protein-RNA interactions in cell-free systems. We found that both ZBP1 and HuD bind the zipcode element in rat β-actin mRNA's 3' UTR. Differences between HuD and ZBP1 were observed in their binding preference to the element. HuD showed a binding preference for U-rich sequence. In contrast, ZBP1 binding to the zipcode RNA depended more on the structural level, as it required the proper spatial organization of a stem-loop that is mainly determined by the U-rich element juxtaposed to the 3' end of a 5'-ACACCC-3' motif. On the basis of this work, we propose that ZBP1 and HuD bind to overlapping sites in the β-actin zipcode, but they recognize different features of this target sequence.

Description: FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with K d app values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners.

Description: Small RNAs, between 18nt and 30nt in length, are a diverse class of non-coding RNAs that mediate a range of cellular processes, from gene regulation to pathogen defense. They guide ribonucleoprotein complexes to their target nucleic acids by Watson–Crick base pairing. We report here that current techniques for small RNA detection and library generation are biased by formation of RNA duplexes. To address this problem, we established FDF-PAGE (fully-denaturing formaldehyde polyacrylamide gel electrophoresis) to prevent annealing of sRNAs to their complement. By applying FDF-PAGE, we provide evidence that both strands of viral small RNA are present in near equimolar ratios, indicating that the predominant precursor is a long double-stranded RNA. Comparing non-denaturing conditions to FDF-PAGE uncovered extensive sequestration of miRNAs in model organisms and allowed us to identify candidate small RNAs under the control of competing endogenous RNAs (ceRNAs). By revealing the full repertoire of small RNAs, we can begin to create a better understanding of small RNA mediated interactions.

Description: Characterization and standardization of inducible transcriptional regulators has transformed how scientists approach biology by allowing precise and tunable control of gene expression. Despite their utility, only a handful of well-characterized regulators exist, limiting the complexity of engineered biological systems. We apply a characterization pipeline to four genetically encoded sensors that respond to acrylate, glucarate, erythromycin and naringenin. We evaluate how the concentration of the inducing chemical relates to protein expression, how the extent of induction affects protein expression kinetics, and how the activation behavior of single cells relates to ensemble measurements. We show that activation of each sensor is orthogonal to the other sensors, and to other common inducible systems. We demonstrate independent control of three fluorescent proteins in a single cell, chemically defining eight unique transcriptional states. To demonstrate biosensor utility in metabolic engineering, we apply the glucarate biosensor to monitor product formation in a heterologous glucarate biosynthesis pathway and identify superior enzyme variants. Doubling the number of well-characterized inducible systems makes more complex synthetic biological circuits accessible. Characterizing sensors that transduce the intracellular concentration of valuable metabolites into fluorescent readouts enables high-throughput screening of biological catalysts and alleviates the primary bottleneck of the metabolic engineering design-build-test cycle.

Description: The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5'-3' exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations.

Description: Ty1 Gag comprises the capsid of virus-like particles and provides nucleic acid chaperone (NAC) functions during retrotransposition in budding yeast. A subgenomic Ty1 mRNA encodes a truncated Gag protein (p22) that is cleaved by Ty1 protease to form p18. p22/p18 strongly inhibits transposition and can be considered an element-encoded restriction factor. Here, we show that only p22 and its short derivatives restrict Ty1 mobility whereas other regions of GAG inhibit mobility weakly if at all. Mutational analyses suggest that p22/p18 is synthesized from either of two closely spaced AUG codons. Interestingly, AUG1p18 and AUG2p18 proteins display different properties, even though both contain a region crucial for RNA binding and NAC activity. AUG1p18 shows highly reduced NAC activity but specific binding to Ty1 RNA, whereas AUG2p18 shows the converse behavior. p22/p18 affects RNA encapsidation and a mutant derivative defective for RNA binding inhibits the RNA chaperone activity of the C-terminal region (CTR) of Gag-p45. Moreover, affinity pulldowns show that p18 and the CTR interact. These results support the idea that one aspect of Ty1 restriction involves inhibition of Gag-p45 NAC functions by p22/p18-Gag interactions.

Description: The reduced expression levels and functional impairment of global miRNAs are related to various human diseases, including cancers. However, relatively little is known about how global miRNA function may be upregulated. Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors. The regulation of miRNA function by ROCK inhibitors is mediated, at least in part, by poly(A)-binding protein-interacting protein 2 (PAIP2), which enhances poly(A)-shortening of miRNA-targeted mRNAs and leads to global upregulation of miRNA function. In the presence of a ROCK inhibitor, PAIP2 expression is enhanced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) through increased ROCK1 nuclear localization and enhanced ROCK1 association with HNF4A. Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription. ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.

Description: Mammalian nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that degrades mRNAs containing premature translation termination codons. Phosphorylation of the essential NMD effector UPF1 by the phosphoinositide-3-kinase-like kinase (PIKK) SMG-1 is a key step in NMD and occurs when SMG-1, its two regulatory factors SMG-8 and SMG-9, and UPF1 form a complex at a terminating ribosome. Electron cryo-microscopy of the SMG-1–8–9-UPF1 complex shows the head and arm architecture characteristic of PIKKs and reveals different states of UPF1 docking. UPF1 is recruited to the SMG-1 kinase domain and C-terminal insertion domain, inducing an opening of the head domain that provides access to the active site. SMG-8 and SMG-9 interact with the SMG-1 C-insertion and promote high-affinity UPF1 binding to SMG-1–8–9, as well as decelerated SMG-1 kinase activity and enhanced stringency of phosphorylation site selection. The presence of UPF2 destabilizes the SMG-1–8–9-UPF1 complex leading to substrate release. Our results suggest an intricate molecular network of SMG-8, SMG-9 and the SMG-1 C-insertion domain that governs UPF1 substrate recruitment and phosphorylation by SMG-1 kinase, an event that is central to trigger mRNA decay.

Description: Model evaluation is a necessary step for better prediction and design of 3D RNA structures. For proteins, this has been widely studied and the knowledge-based statistical potential has been proved to be one of effective ways to solve this problem. Currently, a few knowledge-based statistical potentials have also been proposed to evaluate predicted models of RNA tertiary structures. The benchmark tests showed that they can identify the native structures effectively but further improvements are needed to identify near-native structures and those with non-canonical base pairs. Here, we present a novel knowledge-based potential, 3dRNAscore, which combines distance-dependent and dihedral-dependent energies. The benchmarks on different testing datasets all show that 3dRNAscore are more efficient than existing evaluation methods in recognizing native state from a pool of near-native states of RNAs as well as in ranking near-native states of RNA models.

Description: Ribosomal ribonucleic acid (RNA), transfer RNA and other biological or synthetic RNA polymers can contain nucleotides that have been modified by the addition of chemical groups. Traditional Sanger sequencing methods cannot establish the chemical nature and sequence of these modified-nucleotide containing oligomers. Mass spectrometry (MS) has become the conventional approach for determining the nucleotide composition, modification status and sequence of modified RNAs. Modified RNAs are analyzed by MS using collision-induced dissociation tandem mass spectrometry (CID MS/MS), which produces a complex dataset of oligomeric fragments that must be interpreted to identify and place modified nucleosides within the RNA sequence. Here we report the development of RoboOligo, an interactive software program for the robust analysis of data generated by CID MS/MS of RNA oligomers. There are three main functions of RoboOligo: (i) automated de novo sequencing via the local search paradigm. (ii) Manual sequencing with real-time spectrum labeling and cumulative intensity scoring. (iii) A hybrid approach, coined ‘variable sequencing’, which combines the user intuition of manual sequencing with the high-throughput sampling of automated de novo sequencing.

Description: Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity.

Description: Riboswitches are RNA sensors that regulate gene expression in response to binding of small molecules. Although they conceptually represent simple on/off switches and, therefore, hold great promise for biotechnology and future synthetic biology applications, the induction of gene expression by natural riboswitches after ligand addition or removal is often only moderate and, consequently, the achievable expression levels are not very high. Here, we have designed an RNA amplification-based system that strongly improves the efficiency of riboswitches. We have successfully implemented the method in a biological system for which currently no efficient endogenous tools for inducible (trans)gene expression are available: the chloroplasts of higher plants. We further show that an HIV antigen whose constitutive expression from the chloroplast genome is deleterious to the plant can be inducibly expressed under the control of the R NA amp lification- e nhanced r iboswitch (RAmpER) without causing a mutant phenotype, demonstrating the potential of the method for the production of proteins and metabolites that are toxic to the host cell.

Description: Identification of transcription units (TUs) encoded in a bacterial genome is essential to elucidation of transcriptional regulation of the organism. To gain a detailed understanding of the dynamically composed TU structures, we have used four strand-specific RNA-seq (ssRNA-seq) datasets collected under two experimental conditions to derive the genomic TU organization of Clostridium thermocellum using a machine-learning approach. Our method accurately predicted the genomic boundaries of individual TUs based on two sets of parameters measuring the RNA-seq expression patterns across the genome: expression-level continuity and variance. A total of 2590 distinct TUs are predicted based on the four RNA-seq datasets. Among the predicted TUs, 44% have multiple genes. We assessed our prediction method on an independent set of RNA-seq data with longer reads. The evaluation confirmed the high quality of the predicted TUs. Functional enrichment analyses on a selected subset of the predicted TUs revealed interesting biology. To demonstrate the generality of the prediction method, we have also applied the method to RNA-seq data collected on Escherichia coli and achieved high prediction accuracies. The TU prediction program named SeqTU is publicly available at https://code.google.com/p/seqtu/ . We expect that the predicted TUs can serve as the baseline information for studying transcriptional and post-transcriptional regulation in C. thermocellum and other bacteria.

Description: Detecting genetic variation is one of the main applications of high-throughput sequencing, but is still challenging wherever aligning short reads poses ambiguities. Current state-of-the-art variant calling approaches avoid such regions, arguing that it is necessary to sacrifice detection sensitivity to limit false discovery. We developed a method that links candidate variant positions within repetitive genomic regions into clusters. The technique relies on a resource, a thesaurus of genetic variation, that enumerates genomic regions with similar sequence. The resource is computationally intensive to generate, but once compiled can be applied efficiently to annotate and prioritize variants in repetitive regions. We show that thesaurus annotation can reduce the rate of false variant calls due to mappability by up to three orders of magnitude. We apply the technique to whole genome datasets and establish that called variants in low mappability regions annotated using the thesaurus can be experimentally validated. We then extend the analysis to a large panel of exomes to show that the annotation technique opens possibilities to study variation in hereto hidden and under-studied parts of the genome.

Description: Quadruplex nucleic acids can be formed at the ends of eukaryotic chromosomes. Their formation and stabilisation by appropriate small molecules can be used as a means of inhibiting the telomere maintenance functions of telomerase in human cancer cells. The crystal structures have been determined for a number of complexes between these small molecules and human telomeric DNA and RNA quadruplexes. The detailed structural characteristics of these complexes have been surveyed here and the variations in conformation for the TTA and UUA loops have been explored. Loop conformations have been classified in terms of a number of discrete types and their distribution among the crystal structures. Sugar conformation and backbone angles have also been examined and trends highlighted. One particular loop class has been found to be most prevalent. Implications for in particular, rational drug design, are discussed.

Description: It is common knowledge that conserved residues evolve slowly. We challenge generality of this central tenet of molecular biology by describing the fast evolution of a conserved nucleotide position that is located in the overlap of two open reading frames (ORFs) of polyomaviruses. The de novo ORF is expressed through either the ALTO protein or the Middle T antigen (MT/ALTO), while the ancestral ORF encodes the N-terminal domain of helicase-containing Large T (LT) antigen. In the latter domain the conserved Cys codon of the LXCXE pRB-binding motif constrains codon evolution in the overlapping MT/ALTO ORF to a binary choice between Val and Ala codons, termed here as codon-constrained Val-Ala (COCO-VA) toggling. We found the rate of COCO-VA toggling to approach the speciation rate and to be significantly accelerated compared to the baseline rate of chance substitution in a large monophyletic lineage including all viruses encoding MT/ALTO and three others. Importantly, the COCO-VA site is located in a short linear motif (SLiM) of an intrinsically disordered region, a typical characteristic of adaptive responders. These findings provide evidence that the COCO-VA toggling is under positive selection in many polyomaviruses, implying its critical role in interspecific adaptation, which is unprecedented for conserved residues.