ALBERT

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: 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: A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species. To this end, we developed a multidimensional chromatographic platform to resolve, isolate and quantify all canonical ncRNAs in a single sample of cells or tissue, as well as novel ncRNA species. The applicability of the platform is demonstrated in analyses of ncRNA from bacteria, human cells and plasmodium-infected reticulocytes, as well as a viral RNA genome. Among the many potential applications of this platform are a system-level analysis of the dozens of modified ribonucleosides in ncRNA, characterization of novel long ncRNA species, enhanced detection of rare transcript variants and analysis of viral genomes.

Description: RNA sequencing (RNA-Seq) is a powerful tool for analyzing the identity of cellular RNAs but is often limited by the amount of material available for analysis. In spite of extensive efforts employing existing protocols, we observed that it was not possible to obtain useful sequencing libraries from nuclear RNA derived from cultured human cells after crosslinking and immunoprecipitation (CLIP). Here, we report a method for obtaining strand-specific small RNA libraries for RNA sequencing that requires picograms of RNA. We employ an intramolecular circularization step that increases the efficiency of library preparation and avoids the need for intermolecular ligations of adaptor sequences. Other key features include random priming for full-length cDNA synthesis and gel-free library purification. Using our method, we generated CLIP-Seq libraries from nuclear RNA that had been UV-crosslinked and immunoprecipitated with anti-Argonaute 2 (Ago2) antibody. Computational protocols were developed to enable analysis of raw sequencing data and we observe substantial differences between recognition by Ago2 of RNA species in the nucleus relative to the cytoplasm. This RNA self-circularization approach to RNA sequencing (RC-Seq) allows data to be obtained using small amounts of input RNA that cannot be sequenced by standard methods.

Description: N 6 -methyladenosine (m 6 A) is a prevalent RNA methylation modification involved in the regulation of degradation, subcellular localization, splicing and local conformation changes of RNA transcripts. High-throughput experiments have demonstrated that only a small fraction of the m 6 A consensus motifs in mammalian transcriptomes are modified. Therefore, accurate identification of RNA m 6 A sites becomes emergently important. For the above purpose, here a computational predictor of mammalian m 6 A site named SRAMP is established. To depict the sequence context around m 6 A sites, SRAMP combines three random forest classifiers that exploit the positional nucleotide sequence pattern, the K-nearest neighbor information and the position-independent nucleotide pair spectrum features, respectively. SRAMP uses either genomic sequences or cDNA sequences as its input. With either kind of input sequence, SRAMP achieves competitive performance in both cross-validation tests and rigorous independent benchmarking tests. Analyses of the informative features and overrepresented rules extracted from the random forest classifiers demonstrate that nucleotide usage preferences at the distal positions, in addition to those at the proximal positions, contribute to the classification. As a public prediction server, SRAMP is freely available at http://www.cuilab.cn/sramp/ .

Description: The cyanobacterial hsp17 ribonucleicacid thermometer (RNAT) is one of the smallest naturally occurring RNAT. It forms a single hairpin with an internal 1 x 3-bulge separating the start codon in stem I from the ribosome binding site (RBS) in stem II. We investigated the temperature-dependent regulation of hsp17 by mapping individual base-pair stabilities from solvent exchange nuclear magnetic resonance (NMR) spectroscopy. The wild-type RNAT was found to be stabilized by two critical CG base pairs (C14-G27 and C13-G28). Replacing the internal 1 x 3 bulge by a stable CG base pair in hsp17 rep significantly increased the global stability and unfolding cooperativity as evidenced by circular dichroism spectroscopy. From the NMR analysis, remote stabilization and non-nearest neighbour effects exist at the base-pair level, in particular for nucleotide G28 (five nucleotides apart from the side of mutation). Individual base-pair stabilities are coupled to the stability of the entire thermometer within both the natural and the stabilized RNATs by enthalpy–entropy compensation presumably mediated by the hydration shell. At the melting point the Gibbs energies of the individual nucleobases are equalized suggesting a consecutive zipper-type unfolding mechanism of the RBS leading to a dimmer-like function of hsp17 and switch-like regulation behaviour of hsp17 rep . The data show how minor changes in the nucleotide sequence not only offset the melting temperature but also alter the mode of temperature sensing. The cyanobacterial thermosensor demonstrates the remarkable adjustment of natural RNATs to execute precise temperature control.

Description: Diverse life forms are driven by the evolution of gene regulatory programs including changes in regulator proteins and cis -regulatory elements. Alterations of cis -regulatory elements are likely to dominate the evolution of the gene regulatory networks, as they are subjected to smaller selective constraints compared with proteins and hence may evolve quickly to adapt the environment. Prior studies on cis -regulatory element evolution focus primarily on sequence substitutions of known transcription factor-binding motifs. However, evolutionary models for the dynamics of motif occurrence are relatively rare, and comprehensive characterization of the evolution of all possible motif sequences has not been pursued. In the present study, we propose an algorithm to estimate the strength of purifying selection of a motif sequence based on an evolutionary model capturing the birth and death of motif occurrences on promoters. We term this measure as the ‘evolutionary retention coefficient’, as it is related yet distinct from the canonical definition of selection coefficient in population genetics. Using this algorithm, we estimate and report the evolutionary retention coefficients of all possible 10-nucleotide sequences from the aligned promoter sequences of 27 748. orthologous gene families in 34 mammalian species. Intriguingly, the evolutionary retention coefficients of motifs are intimately associated with their functional relevance. Top-ranking motifs (sorted by evolutionary retention coefficients) are significantly enriched with transcription factor-binding sequences according to the curated knowledge from the TRANSFAC database and the ChIP-seq data generated from the ENCODE Consortium. Moreover, genes harbouring high-scoring motifs on their promoters retain significantly coherent expression profiles, and those genes are over-represented in the functional classes involved in gene regulation. The validation results reveal the dependencies between natural selection and functions of cis -regulatory elements and shed light on the evolution of gene regulatory networks.

Description: Mirtrons are a recently described category of microRNA (miRNA) relying on splicing rather than processing by the microprocessor complex to generate pre-miRNA precursors of the RNA interference (RNAi) pathway. Their discovery and subsequent verification provides important information about a distinct class of miRNA and inherent advantages that could be exploited to silence genes of interest. These include micro-processor-independent biogenesis, pol-II-dependent transcription, accurate species generation and the delivery of multiple artificial mirtrons as introns within a single host transcript. Here we determined the sequence motifs required for correct processing of the mmu-miR-1224 mirtron and incorporated these into artificial mirtrons targeting Parkinson’s disease-associated LRRK2 and α-synuclein genes. By incorporating these rules associated with processing and splicing, artificial mirtrons could be designed and made to silence complementary targets either at the mRNA or protein level. We further demonstrate with a LRRK2 targeting artificial mirtron that neuronal-specific silencing can be directed under the control of the human synapsin promoter. Finally, multiple mirtrons were co-delivered within a single host transcript, an eGFP reporter, to allow simultaneous targeting of two or more targets in a combinatorial approach. Thus, the unique characteristics of artificial mirtrons make this an attractive approach for future RNAi applications.

Description: Despite the many advantages of Caenorhabditis elegans , biochemical approaches to study tissue-specific gene expression in post-embryonic stages are challenging. Here, we report a novel experimental approach for efficient determination of tissue-specific transcriptomes involving the rapid release and purification of nuclei from major tissues of post-embryonic animals by f luorescence- a ctivated n uclei s orting (FANS), followed by deep sequencing of linearly amplified 3'-end regions of transcripts (3'-end-seq). We employed these approaches to compile the transcriptome of the developed C. elegans intestine and used this to analyse tissue-specific cleavage and polyadenylation. In agreement with intestinal-specific gene expression, highly expressed genes have enriched GATA-elements in their promoter regions and their functional properties are associated with processes that are characteristic for the intestine. We systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including more than 3000 sites that have previously not been identified. The detailed analysis of the 3'-ends of the nuclear mRNA revealed widespread alternative polyA site use (APA) in intestinally expressed genes. Importantly, we found that intestinal polyA sites that undergo APA tend to have U-rich and/or A-rich upstream auxiliary elements that may contribute to the regulation of 3'-end formation in the intestine.

Description: A novel biosensing approach for the label-free detection of nucleic acid sequences of short and large lengths has been implemented, with special emphasis on targeting RNA sequences with secondary structures. The approach is based on selecting 8-aminoadenine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. With this strategy, we have detected short DNA sequences (32-mer) and purified RNA (103-mer) at the femtomol level in a few minutes in an easy and level-free way. This approach is particularly suitable for the detection of RNA molecules with predicted secondary structures, reaching a limit of detection of 50 fmol without any label or amplification steps. Our methodology has shown a marked enhancement for the detection (18% for short DNA and 54% for RNA), when compared with the conventional duplex approach, highlighting the large difficulty of the duplex approach to detect nucleic acid sequences, especially those exhibiting stable secondary structures. We believe that our strategy could be of great interest to the RNA field.

Description: A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop–receptor interaction in magnesium and potassium solutions. Both the kinetics and thermodynamics were obtained in individual ITC experiments, and analysis of the kinetic data over a range of temperatures provided Arrhenius activation energies ( H ) and Eyring transition state entropies ( S ). The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium. In potassium, association is highly exothermic ( H 25°C = –41.6 ± 1.2 kcal/mol in 150 mM KCl) and the transition state is enthalpically barrierless ( H = –0.6 ± 0.5). These parameters are sigificantly positively shifted in magnesium ( H 25°C = –20.5 ± 2.1 kcal/mol, H = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl 2 ). Mixed salt solutions approximating physiological conditions exhibit an intermediate thermodynamic character. The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.

Description: Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant ( k cat ) of a model ribozyme.

Description: Compared to transcriptional activation, other mechanisms of gene regulation have not been widely exploited for the control of transgenes. One barrier to the general use and application of alternative splicing is that splicing-regulated transgenes have not been shown to be reliably and simply designed. Here, we demonstrate that a cassette bearing a suicide exon can be inserted into a variety of open reading frames (ORFs), generating transgenes whose expression is activated by exon skipping in response to a specific protein inducer. The surprisingly minimal sequence requirements for the maintenance of splicing fidelity and regulation indicate that this splicing cassette can be used to regulate any ORF containing one of the amino acids Glu, Gln or Lys. Furthermore, a single copy of the splicing cassette was optimized by rational design to confer robust gene activation with no background expression in plants. Thus, conditional splicing has the potential to be generally useful for transgene regulation.

Description: Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a U A G premature termination codon (PTC) to tryptophan (U G G) was improved from ~11 % to ~70 %. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool.

Description: RNA 2'-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from Bacteria, Archaea and Eukarya. RNAs bearing 2'-O-methylations show increased resistance to degradation and enhanced stability in helices. While the exact role of each 2'-O-Me residue remained elusive, the catalytic protein Fibrillarin (Nop1 in yeast) responsible for 2'-O-methylation in eukaryotes, is associated with human pathologies. Therefore, there is an urgent need to precisely map and quantify hundreds of 2'-O-Me residues in RNA using high-throughput technologies. Here, we develop a reliable protocol using alkaline fragmentation of total RNA coupled to a commonly used ligation approach, and Illumina sequencing. We describe a methodology to detect 2'-O-methylations with high sensitivity and reproducibility even with limited amount of starting material (1 ng of total RNA). The method provides a quantification of the 2'-O-methylation occupancy of a given site, allowing to detect relatively small changes (〉10%) in 2'-O-methylation profiles. Altogether this technique unlocks a technological barrier since it will be applicable for routine parallel treatment of biological and clinical samples to decipher the functions of 2'-O-methylations in pathologies.

Description: Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional mechanism, evolved to diversify the transcriptome in metazoa. In addition to wide-spread editing in non-coding regions protein recoding by RNA editing allows for fine tuning of protein function. Functional consequences are only known for some editing sites and the combinatorial effect between multiple sites (functional epistasis) is currently unclear. Similarly, the interplay between RNA editing and splicing, which impacts on post-transcriptional gene regulation, has not been resolved. Here, we describe a versatile antisense approach, which will aid resolving these open questions. We have developed and characterized morpholino oligos targeting the most efficiently edited site—the AMPA receptor GluA2 Q/R site. We show that inhibition of editing closely correlates with intronic editing efficiency, which is linked to splicing efficiency. In addition to providing a versatile tool our data underscore the unique efficiency of a physiologically pivotal editing site.

Description: Systematic analysis of the RNA-protein interactome requires robust and scalable methods. We here show the combination of two completely orthogonal, generic techniques to identify RNA-protein interactions: PAR-CLIP reveals a collection of RNAs bound to a protein whereas SILAC-based RNA pull-downs identify a group of proteins bound to an RNA. We investigated binding sites for five different proteins (IGF2BP1-3, QKI and PUM2) exhibiting different binding patterns. We report near perfect agreement between the two approaches. Nevertheless, they are non-redundant, and ideally complement each other to map the RNA-protein interaction network.

Description: RNA snap ™ is a simple and novel method that recovers all intracellular RNA quantitatively (〉99%), faster (〈15 min) and less expensively (~3 cents/sample) than any of the currently available RNA isolation methods. In fact, none of the bacterial RNA isolation methods, including the commercial kits, are effective in recovering all species of intracellular RNAs (76–5700 nt) with equal efficiency, which can lead to biased results in genome-wide studies involving microarray or RNAseq analysis. The RNA snap ™ procedure yields ~60 µg of RNA from 10 8 Escherichia coli cells that can be used directly for northern analysis without any further purification. Based on a comparative analysis of specific transcripts ranging in size from 76 to 5700 nt, the RNA snap ™ method provided the most accurate measure of the relative amounts of the various intracellular RNAs. Furthermore, the RNA snap ™ RNA was successfully used in enzymatic reactions such as RNA ligation, reverse transcription, primer extension and reverse transcriptase–polymerase chain reaction, following sodium acetate/ethanol precipitation. The RNA snap ™ method can be used to isolate RNA from a wide range of Gram-negative and Gram-positive bacteria as well as yeast.

Description: Splicing of human pre-mRNA is reciprocally coupled to 3' end formation by terminal exon definition, which occurs co-transcriptionally. It is required for the final maturation of most human pre-mRNAs and is therefore important to understand. We have used several strategies to block splicing at specific stages in vivo and studied their effect on 3' end formation. We demonstrate that a terminal splice acceptor site is essential to establish coupling with the poly(A) signal in a chromosomally integrated β-globin gene. This is in part to alleviate the suppression of 3' end formation by U1 small nuclear RNA, which is known to bind pre-mRNA at the earliest stage of spliceosome assembly. Interestingly, blocks to splicing that are subsequent to terminal splice acceptor site function, but before catalysis, have little observable effect on 3' end formation. These data suggest that early stages of spliceosome assembly are sufficient to functionally couple splicing and 3' end formation, but that on-going intron removal is less critical.

Description: A large fraction of protein-coding genes in metazoans undergo alternative pre-mRNA splicing in tissue- or cell-type-specific manners. Recent genome-wide approaches have identified many putative-binding sites for some of tissue-specific trans -acting splicing regulators. However, the mechanisms of splicing regulation in vivo remain largely unknown. To elucidate the modes of splicing regulation by the neuron-specific CELF family RNA-binding protein UNC-75 in Caenorhabditis elegans , we performed deep sequencing of poly(A) + RNAs from the unc-75(+)- and unc-75- mutant worms and identified more than 20 cassette and mutually exclusive exons repressed or activated by UNC-75. Motif searches revealed that (G/U)UGUUGUG stretches are enriched in the upstream and downstream introns of the UNC-75-repressed and -activated exons, respectively. Recombinant UNC-75 protein specifically binds to RNA fragments carrying the (G/U)UGUUGUG stretches in vitro . Bi-chromatic fluorescence alternative splicing reporters revealed that the UNC-75-target exons are regulated in tissue-specific and (G/U)UGUUGUG element-dependent manners in vivo . The unc-75 mutation affected the splicing reporter expression specifically in the nervous system. These results indicate that UNC-75 regulates alternative splicing of its target exons in neuron-specific and position-dependent manners through the (G/U)UGUUGUG elements in C. elegans . This study thus reveals the repertoire of target events for the CELF family in the living organism.

Description: Misfolding and associated loss of function are common problems in constructing fusion RNA complexes due to changes in energy landscape and the nearest-neighbor principle. Here we report the incorporation and application of the pRNA-3WJ motif of the phi29 DNA packaging motor into fusion RNA with controllable and predictable folding. The motif included three discontinuous ~18 nucleotide (nt) fragments, displayed a distinct low folding energy (Shu D et al. , Nature Nanotechnology , 2011, 6:658–667), and folded spontaneously into a leading core that enabled the correct folding of other functionalities fused to the RNA complex. Three individual fragments dispersed at any location within the sequence allowed the other RNA functional modules to fold into their original structures with authentic functions, as tested by Hepatitis B virus ribozyme, siRNA, and aptamers for malachite green (MG), spinach, and streptavidin (STV). Only nine complementary nucleotides were present for any two of the three ~18-nt fragments, but the three 9 bp branches were so powerful that they disrupted other double strands with more than 15 bp within the fusion RNA. This system enabled the production of fusion complexes harboring multiple RNA functionalities with correct folding for potential applications in biotechnology, nanomedicine and nanotechnology. We also applied this system to investigate the principles governing the folding of RNA in vivo and in vitro . Temporal production of RNA sequences during in vivo transcription caused RNA to fold into different conformations that could not be predicted with routine principles derived from in vitro studies.

Description: The substitution of 2'-fluoro for 2'-hydroxyl moieties in RNA substantially improves the stability of RNA. RNA stability is a major issue in RNA research and applications involving RNA. We report that the RNA polymerase from the marine cyanophage Syn5 has an intrinsic low discrimination against the incorporation of 2'-fluoro dNMPs during transcription elongation. The presence of both magnesium and manganese ions at high concentrations further reduce this discrimination without decreasing the efficiency of incorporation. We have constructed a Syn5 RNA polymerase in which tyrosine 564 is replaced with phenylalanine (Y564F) that further decreases the discrimination against 2'-fluoro-dNTPs during RNA synthesis. Sequence elements in DNA templates that affect the yield of RNA and incorporation of 2'-fluoro-dNMPs by Syn5 RNA polymerase have been identified.

Description: Recent studies have revealed significant roles of RNA structure in almost every step of RNA processing, including transcription, splicing, transport and translation. RNase footprint sequencing (RNase-seq) has emerged to dissect RNA structures at the genome scale. However, it remains challenging to analyze RNase-seq data because of the issues of signal sparsity, variability and correlations among various RNases. We present a probabilistic framework, joint Poisson-gamma mixture (JPGM), for integrative modeling of multiple RNase-seq profiles. Combining JPGM with hidden Markov model allows genome-wide inference of RNA structures. We apply the joint modeling approach for inferring base pairing states on simulated data sets and RNase-seq profiles of the double-strand specific RNase V1 and single-strand specific RNase S1 in yeast. We demonstrate that joint analysis of V1 and S1 profiles outputs interpretable RNA structure states, while approaches that analyze each profile separately do not. The joint modeling approach predicts the structure states of all nucleotides in 3196 transcripts of yeast without compromising accuracy, while the simple thresholding approach misses 43% of the nucleotides. Furthermore, the posterior probabilities outputted by our model are able to resolve the structural ambiguity of 300 000 nucleotides with overlapping V1 and S1 cleavage sites. Our model also generates RNA accessibilities, which are associated with three-dimensional conformations.