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: RNA sequencing (RNAseq) has become the method of choice for transcriptome analysis, yet no consensus exists as to the most appropriate pipeline for its analysis, with current benchmarks suffering important limitations. Here, we address these challenges through a rich benchmarking resource harnessing (i) two RNAseq datasets including ERCC ExFold spike-ins; (ii) Nanostring measurements of a panel of 150 genes on the same samples; (iii) a set of internal, genetically-determined controls; (iv) a reanalysis of the SEQC dataset; and (v) a focus on relative quantification (i.e. across-samples). We use this resource to compare different approaches to each step of RNAseq analysis, from alignment to differential expression testing. We show that methods providing the best absolute quantification do not necessarily provide good relative quantification across samples, that count-based methods are superior for gene-level relative quantification, and that the new generation of pseudo-alignment-based software performs as well as established methods, at a fraction of the computing time. We also assess the impact of library type and size on quantification and differential expression analysis. Finally, we have created a R package and a web platform to enable the simple and streamlined application of this resource to the benchmarking of future methods.

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: Cap analysis of gene expression (CAGE) is a high-throughput method for transcriptome analysis that provides a single base-pair resolution map of transcription start sites (TSS) and their relative usage. Despite their high resolution and functional significance, published CAGE data are still underused in promoter analysis due to the absence of tools that enable its efficient manipulation and integration with other genome data types. Here we present CAGEr , an R implementation of novel methods for the analysis of differential TSS usage and promoter dynamics, integrated with CAGE data processing and promoterome mining into a first comprehensive CAGE toolbox on a common analysis platform. Crucially, we provide collections of TSSs derived from most published CAGE datasets, as well as direct access to FANTOM5 resource of TSSs for numerous human and mouse cell/tissue types from within R, greatly increasing the accessibility of precise context-specific TSS data for integrative analyses. The CAGEr package is freely available from Bioconductor at http://www.bioconductor.org/packages/release/bioc/html/CAGEr.html .

Description: MicroRNAs (miRNAs), small non-coding RNA molecules, are important biomarkers for research and medical purposes. Here, we describe the development of a fast and simple method using highly fluorescent oligonucleotide-silver nanocluster probes (DNA/AgNCs) to efficiently detect specific miRNAs. Due to the great sequence diversity of miRNAs in humans and other organisms, a uniform strategy for miRNA detection is attractive. The concept presented is an oligonucleotide-based locking-to-unlocking system that can be endowed with miRNA complementarity while maintaining the same secondary structure. The locking-to-unlocking system is based on fold-back anchored DNA templates that consist of a cytosine-rich loop for AgNCs stabilization, an miRNA recognition site and an overlap region for hairpin stabilization. When an miRNA is recognized, fluorescence in the visible region is specifically extinguished in a concentration-dependent manner. Here, the exact composition of the fold-back anchor for the locking-to-unlocking system has been systematically optimized, balancing propensity for loop-structure formation, encapsulation of emissive AgNCs and target sensitivity. It is demonstrated that the applied strategy successfully can detect a number of cancer related miRNAs in RNA extracts from human cancer cell lines.

Description: The variability and complexity of the transcription initiation process was examined by adapting RNA ligase-mediated rapid amplification of 5' cDNA ends (5'-RACE) to Next-Generation Sequencing (NGS). We oligo-labelled 5'-m 7 G-capped mRNA from two genes, the simple mono-exonic Beta-2-Adrenoceptor (ADRB2R) and the complex multi-exonic Glucocorticoid Receptor (GR, NR3C1) , and detected a variability in TSS location that has received little attention up to now. Transcription was not initiated at a fixed TSS, but from loci of 4 to 10 adjacent nucleotides. Individual TSSs had frequencies from 〈0.001% to 38.5% of the total gene-specific 5' m 7 G-capped transcripts. ADRB2R used a single locus consisting of 4 adjacent TSSs. Unstimulated, the GR used a total of 358 TSSs distributed throughout 38 loci, that were principally in the 5' UTRs and were spliced using established donor and acceptor sites. Complete demethylation of the epigenetically sensitive GR promoter with 5-azacytidine induced one new locus and 127 TSSs, 12 of which were unique. We induced GR transcription with dexamethasone and Interferon-, adding one new locus and 185 additional TSSs distributed throughout the promoter region. In-vitro the TSS microvariability regulated mRNA translation efficiency and the relative abundance of the different GR N-terminal protein isoform levels.

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: Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2'OMe-RNA probes. The HCR northern blot protocol takes ~1.5 days independent of the number of target RNAs.

Description: Although RNA-Seq data provide unprecedented isoform-level expression information, detection of alternative isoform regulation (AIR) remains difficult, particularly when working with an incomplete transcript annotation. We introduce JunctionSeq, a new method that builds on the statistical techniques used by the well-established DEXSeq package to detect differential usage of both exonic regions and splice junctions. In particular, JunctionSeq is capable of detecting differential usage of novel splice junctions without the need for an additional isoform assembly step, greatly improving performance when the available transcript annotation is flawed or incomplete. JunctionSeq also provides a powerful and streamlined visualization toolset that allows bioinformaticians to quickly and intuitively interpret their results. We tested our method on publicly available data from several experiments performed on the rat pineal gland and Toxoplasma gondii , successfully detecting known and previously validated AIR genes in 19 out of 19 gene-level hypothesis tests. Due to its ability to query novel splice sites, JunctionSeq is still able to detect these differences even when all alternative isoforms for these genes were not included in the transcript annotation. JunctionSeq thus provides a powerful method for detecting alternative isoform regulation even with low-quality annotations. An implementation of JunctionSeq is available as an R/Bioconductor package.

Description: Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii , and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine–rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii . Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes.

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: Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized 〉300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts.

Description: As the use of RNA-seq has popularized, there is an increasing consciousness of the importance of experimental design, bias removal, accurate quantification and control of false positives for proper data analysis. We introduce the NOISeq R-package for quality control and analysis of count data. We show how the available diagnostic tools can be used to monitor quality issues, make pre-processing decisions and improve analysis. We demonstrate that the non-parametric NOISeqBIO efficiently controls false discoveries in experiments with biological replication and outperforms state-of-the-art methods. NOISeq is a comprehensive resource that meets current needs for robust data-aware analysis of RNA-seq differential expression.

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: Methods that permit controlled changes in the expression of genes are important tools for biological and medical research, and for biotechnological applications. Conventional methods are directed at individually changing each gene, its regulatory elements or its mRNA's translation rate. We demonstrate that the CRISPR-associated DNA-binding Cascade complex can be used for efficient, long-lasting and programmable gene silencing. When Cascade is targeted to a promoter sequence the transcription of the downstream gene is inhibited, resulting in dramatically reduced expression. The specificity of Cascade binding is provided by the integral crRNA component, which is easily designed to target virtually any stretch of DNA. Cascade targeted to the ORF sequence of the gene can also silence expression, albeit at lower efficiency. The system can be used to silence plasmid and chromosome targets, simultaneously target several genes and is active in different bacterial species and strains. The findings described here are an addition to the expanding range of CRISPR-based technologies and may be adapted to additional organisms and cell systems.

Description: Regardless of the advent of high-throughput sequencing, microarrays remain central in current biomedical research. Conventional microarray analysis pipelines apply data reduction before the estimation of differential expression, which is likely to render the estimates susceptible to noise from signal summarization and reduce statistical power. We present a probe-level framework, which capitalizes on the high number of concurrent measurements to provide more robust differential expression estimates. The framework naturally extends to various experimental designs and target categories (e.g. transcripts, genes, genomic regions) as well as small sample sizes. Benchmarking in relation to popular microarray and RNA-sequencing data-analysis pipelines indicated high and stable performance on the Microarray Quality Control dataset and in a cell-culture model of hypoxia. Experimental-data-exhibiting long-range epigenetic silencing of gene expression was used to demonstrate the efficacy of detecting differential expression of genomic regions, a level of analysis not embraced by conventional workflows. Finally, we designed and conducted an experiment to identify hypothermia-responsive genes in terms of monotonic time-response. As a novel insight, hypothermia-dependent up-regulation of multiple genes of two major antioxidant pathways was identified and verified by quantitative real-time PCR.

Description: Computational techniques have been used to design a novel class of RNA architecture with expected improved resistance to nuclease degradation, while showing interference RNA activity. The in silico designed structure consists of a 24–29 bp duplex RNA region linked on both ends by N-alkyl-N dimeric nucleotides (BCn dimers; n = number of carbon atoms of the alkyl chain). A series of N-alkyl-N capped dumbbell-shaped structures were efficiently synthesized by double ligation of BCn-loop hairpins. The resulting BCn-loop dumbbells displayed experimentally higher biostability than their 3'-N-alkyl-N linear version, and were active against a range of mRNA targets. We studied first the effect of the alkyl chain and stem lengths on RNAi activity in a screen involving two series of dumbbell analogues targeting Renilla and Firefly luciferase genes. The best dumbbell design (containing BC6 loops and 29 bp) was successfully used to silence GRB7 expression in HER2+ breast cancer cells for longer periods of time than natural siRNAs and known biostable dumbbells. This BC6-loop dumbbell-shaped structure displayed greater anti-proliferative activity than natural siRNAs.

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: mRNA expression involves transcription initiation, elongation and degradation. In cells, these dynamic processes are highly regulated. However, experimental characterization of the dynamic processes in vivo is difficult due to the paucity of methods capable of direct measurements. We present a highly sensitive and versatile method enabling direct characterization of the dynamic processes. Our method is based on single-molecule fluorescence in situ hybridization (smFISH) and quantitative analyses of hybridization signals. We hybridized multiple probes labelled with spectrally distinct fluorophores to multiple sub-regions of single mRNAs, and visualized the kinetics of synthesis and degradation of the sub-regions. Quantitative analyses of the data lead to absolute quantification of the lag time of mRNA induction (the time it takes for external signals to activate transcription initiation), transcription initiation rate, transcription elongation speed (i.e. mRNA chain-growth speed), the rate of premature termination of transcripts and degradation rates. Applying our method to three different biological problems, we demonstrated how our method may be applicable to reveal dynamics of mRNA expression that was difficult to study previously. We expect such absolute quantification can greatly facilitate understanding of gene expression and its regulation working at the levels of transcriptional initiation, elongation and degradation.

Description: Current approaches to profiling tissue-specific gene expression in C. elegans require delicate manipulation and are difficult under certain conditions, e.g. from dauer or aging worms. We have developed an easy and robust method for tissue-specific RNA-seq by taking advantage of the endogenous trans-splicing process. In this method, transgenic worms are generated in which a spliced leader (SL) RNA gene is fused with a sequence tag and driven by a tissue-specific promoter. Only in the tissue of interest, the tagged SL RNA gene is transcribed and then trans-spliced onto mRNAs. The tag allows enrichment and sequencing of mRNAs from that tissue only. As a proof of principle, we profiled the muscle transcriptome, which showed high coverage and efficient enrichment of muscle specific genes, with low background noise. To demonstrate the robustness of our method, we profiled muscle gene expression in dauer larvae and aging worms, revealing gene expression changes consistent with the physiology of these stages. The resulting muscle transcriptome also revealed 461 novel RNA transcripts, likely muscle-expressed long non-coding RNAs. In summary, the splicing-based RNA tagging (SRT) method provides a convenient and robust tool to profile trans-spliced genes and identify novel transcripts in a tissue-specific manner, with a low false positive rate.

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: The use of alternative poly(A) sites is common and affects the post-transcriptional fate of mRNA, including its stability, subcellular localization and translation. Here, we present a method to identify poly(A) sites in a genome-wide and strand-specific manner. This method, termed 3'T-fill, initially fills in the poly(A) stretch with unlabeled dTTPs, allowing sequencing to start directly after the poly(A) tail into the 3'-untranslated regions (UTR). Our comparative analysis demonstrates that it outperforms existing protocols in quality and throughput and accurately quantifies RNA levels as only one read is produced from each transcript. We use this method to characterize the diversity of polyadenylation in Saccharomyces cerevisiae , showing that alternative RNA molecules are present even in a genetically identical cell population. Finally, we observe that overlap of convergent 3'-UTRs is frequent but sharply limited by coding regions, suggesting factors that restrict compression of the yeast genome.

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.

Description: Myoblasts are precursor skeletal muscle cells that differentiate into fused, multinucleated myotubes. Current single-cell microfluidic methods are not optimized for capturing very large, multinucleated cells such as myotubes. To circumvent the problem, we performed single-nucleus transcriptome analysis. Using immortalized human myoblasts, we performed RNA-seq analysis of single cells (scRNA-seq) and single nuclei (snRNA-seq) and found them comparable, with a distinct enrichment for long non-coding RNAs (lncRNAs) in snRNA-seq. We then compared snRNA-seq of myoblasts before and after differentiation. We observed the presence of mononucleated cells (MNCs) that remained unfused and analyzed separately from multi-nucleated myotubes. We found that while the transcriptome profiles of myoblast and myotube nuclei are relatively homogeneous, MNC nuclei exhibited significant heterogeneity, with the majority of them adopting a distinct mesenchymal state. Primary transcripts for microRNAs (miRNAs) that participate in skeletal muscle differentiation were among the most differentially expressed lncRNAs, which we validated using NanoString. Our study demonstrates that snRNA-seq provides reliable transcriptome quantification for cells that are otherwise not amenable to current single-cell platforms. Our results further indicate that snRNA-seq has unique advantage in capturing nucleus-enriched lncRNAs and miRNA precursors that are useful in mapping and monitoring differential miRNA expression during cellular differentiation.