Hyeshik Chang

Motivation: Understanding the details of protein–RNA interactions is important to reveal the functions of both the RNAs and the proteins. In these interactions, the secondary structures of the RNAs play an important role. Because RNA secondary structures in protein–RNA complexes are variable, considering the ensemble of RNA secondary structures is a useful approach. In particular, recent studies have supported the idea that, in the analysis of RNA secondary structures, the base-pairing probabilities (BPPs) of RNAs (i.e. the probabilities of forming a base pair in the ensemble of RNA secondary structures) provide richer and more robust information about the structures than a single RNA secondary structure, for example, the minimum free energy structure or a snapshot of structures in the Protein Data Bank. However, there has been no investigation of the BPPs in protein–RNA interactions.

Results: In this study, we analyzed BPPs of RNA molecules involved in known protein–RNA complexes in the Protein Data Bank. Our analysis suggests that, in the tertiary structures, the BPPs (which are computed using only sequence information) for unpaired nucleotides with intermolecular hydrogen bonds (hbonds) to amino acids were significantly lower than those for unpaired nucleotides without hbonds. On the other hand, no difference was found between the BPPs for paired nucleotides with and without intermolecular hbonds. Those findings were commonly supported by three probabilistic models, which provide the ensemble of RNA secondary structures, including the McCaskill model based on Turner’s free energy of secondary structures.

Contact: iwakiri@cb.k.u-tokyo.ac.jp or mhamada@cb.k.u-tokyo.ac.jp

Supplementary information: Supplementary data are available at Bioinformatics online.

by Michael Stadler, Andrew Fire

Nematodes of the genus Caenorhabditis enter a developmental diapause state after hatching in the absence of food. To better understand the relative contributions of distinct regulatory modalities to gene expression changes associated with this developmental transition, we characterized genome-wide changes in mRNA abundance and translational efficiency associated with L1 diapause exit in four species using ribosome profiling and mRNA-seq. We found a strong tendency for translational regulation and mRNA abundance processes to act synergistically, together effecting a dramatic remodeling of the gene expression program. While gene-specific differences were observed between species, overall translational dynamics were broadly and functionally conserved. A striking, conserved feature of the response was strong translational suppression of ribosomal protein production during L1 diapause, followed by activation upon resumed development. On a global scale, ribosome footprint abundance changes showed greater similarity between species than changes in mRNA abundance, illustrating a substantial and genome-wide contribution of translational regulation to evolutionary maintenance of stable gene expression.

Mirela Marasovic, Manuel Zocco, Mario Halic. RNAi is a conserved mechanism in which small RNAs induce silencing of complementary targets. We have previously identified priRNAs, a class of Dicer-independent small RNAs in fission yeast. The me....

Mary Anne T. Rubio, Zdeněk Paris, Kirk W. Gaston, Ian M.C. Fleming, Paul Sample, Christopher R. Trotta, Juan D. Alfonzo. In cells, tRNAs are synthesized as precursor molecules bearing extra sequences at their 5′ and 3′ ends. Some tRNAs also contain introns, which, in archaea and eukaryotes, are cleaved by an evoluti....

Bonnie Bassler and Larry Smarr have a gift for enthralling audiences. They share advice on how to make powerful public presentations. Author: Jon Cohen

Genome-wide analysis of A-to-I RNA editing by single-molecule sequencing in Drosophila.

Nat Struct Mol Biol. 2013 Sep 29;

Authors: St Laurent G, Tackett MR, Nechkin S, Shtokalo D, Antonets D, Savva YA, Maloney R, Kapranov P, Lawrence CE, Reenan RA

Abstract
The accurate and thorough genome-wide detection of adenosine-to-inosine editing, a biologically indispensable process, has proven challenging. Here, we present a discovery pipeline in adult Drosophila, with 3,581 high-confidence editing sites identified with an estimated accuracy of 87%. The target genes and specific sites highlight global biological properties and functions of RNA editing, including hitherto-unknown editing in well-characterized classes of noncoding RNAs and 645 sites that cause amino acid substitutions, usually at conserved positions. The spectrum of functions that these gene targets encompass suggests that editing participates in a diverse set of cellular processes. Editing sites in Drosophila exhibit sequence-motif preferences and tend to be concentrated within a small subset of total RNAs. Finally, editing regulates expression levels of target mRNAs and strongly correlates with alternative splicing.

PMID: 24077224 [PubMed - as supplied by publisher]

Article

MicroRNAs are thought to confer robustness to biological processes, but clear experimental evidence is still needed. Here, Siciliano et al . construct a toggle-switch in mammalian cells to show that microRNAs buffer fluctuations in protein levels, thereby providing phenotypic robustness to gene regulatory networks.

Nature Communications doi: 10.1038/ncomms3364

Authors: Velia Siciliano, Immacolata Garzilli, Chiara Fracassi, Stefania Criscuolo, Simona Ventre, Diego di Bernardo

UPF1 is a DNA/RNA helicase with essential roles in nonsense-mediated mRNA decay (NMD) and embryonic development. How UPF1 regulates target abundance and the relationship between NMD and embryogenesis are not well understood. To explore how NMD shapes the embryonic transcriptome, we integrated genome-wide analyses of UPF1 binding locations, NMD-regulated gene expression, and translation in murine embryonic stem cells (mESCs). We identified over 200 direct UPF1 binding targets using crosslinking/immunoprecipitation-sequencing (CLIP-seq) and revealed a repression pathway that involves 3' UTR binding by UPF1 and translation but is independent of canonical targeting features involving 3' UTR length and stop codon placement. Interestingly, NMD targeting of this set of mRNAs occurs in other mouse tissues and is conserved in human. We also show, using ribosome footprint profiling, that actively translated upstream open reading frames (uORFs) are enriched in transcription factor mRNAs and predict mRNA repression by NMD, while poorly translated mRNAs escape repression. Together, our results identify novel NMD determinants and targets and provide context for understanding the impact of UPF1 and NMD on the mESC transcriptome.

MicroRNAs (miRNAs) derive from longer precursors with fold-back structures. While animal miRNA precursors have homogenous structures, plant precursors comprise a collection of fold-backs with variable size and shape. Here, we design an approach to systematically analyze miRNA processing intermediates and characterize the biogenesis of most of the evolutionarily conserved miRNAs present in Arabidopsis thaliana. We found that plant miRNAs are processed by four mechanisms, depending on the sequential direction of the processing machinery and the number of cuts required to release the miRNA. Classification of the precursors according to their processing mechanism revealed specific structural determinants for each group. We found that the complexity of the miRNA processing pathways occurs in both ancient and evolutionarily young sequences and that members of the same family can be processed in different ways. We observed that different structural determinants compete for the processing machinery and that alternative miRNAs can be generated from a single precursor. The results provide an explanation for the structural diversity of miRNA precursors in plants and new insights toward the understanding of the biogenesis of small RNAs.

Pre-mRNA splicing is required for the accurate expression of virtually all human protein coding genes. However, splicing also plays important roles in coordinating subsequent steps of pre-mRNA processing such as polyadenylation and mRNA export. Here, we test the hypothesis that nuclear pre-mRNA processing influences the polyribosome association of alternative mRNA isoforms. By comparing isoform ratios in cytoplasmic and polyribosomal extracts, we determined that the alternative products of ~30% (597/1954) of mRNA processing events are differentially partitioned between these subcellular fractions. Many of the events exhibiting isoform-specific polyribosome association are highly conserved across mammalian genomes, underscoring their possible biological importance. We find that differences in polyribosome association may be explained, at least in part by the observation that alternative splicing alters the cis-regulatory landscape of mRNAs isoforms. For example, inclusion or exclusion of upstream open reading frames (uORFs) in the 5'UTR as well as Alu-elements and microRNA target sites in the 3'UTR have a strong influence on polyribosome association of alternative mRNA isoforms. Taken together, our data demonstrate for the first time the potential link between alternative splicing and translational control of the resultant mRNA isoforms.

Splicing requires a complex set of ATP-dependent macromolecular associations that lead to the rearrangement of just a few covalent bonds in the pre-mRNA substrate. Seeing only the covalent bonds breaking and forming is seeing only a very small part of the process. Analysis of native splicing complexes into which the radiolabeled substrate has been assembled, but not necessarily completely reacted, has provided a good understanding of the process. This protocol describes a gel method for detecting and analyzing yeast splicing complexes formed in vitro on a radiolabeled pre-mRNA substrate. Complexes formed during the splicing reaction are quenched by dilution and addition of an excess of RNA, which is thought to strip nonspecifically bound proteins from the labeled substrate RNA. After loading on a low-percentage, low-cross-linking ratio composite agarose–acrylamide gel (in 10% glycerol), labeled bands are detected. These can be extracted and shown to contain small nuclear RNAs (snRNAs) and partly reacted pre-mRNA.

Factor activating Pos9 (Fap7) is an essential ribosome biogenesis factor important for the assembly of the small ribosomal subunit with an uncommon dual ATPase and adenylate kinase activity. Depletion of Fap7 or mutations in its ATPase motifs lead to defects in small ribosomal subunit rRNA maturation, the absence of ribosomal...

Nature advance online publication 22 September 2013. doi:10.1038/nature12632

Authors: Miler T. Lee, Ashley R. Bonneau, Carter M. Takacs, Ariel A. Bazzini, Kate R. DiVito, Elizabeth S. Fleming & Antonio J. Giraldez

Nature advance online publication 22 September 2013. doi:10.1038/nature12543

Authors: Ulf-Peter Guenther, Lindsay E. Yandek, Courtney N. Niland, Frank E. Campbell, David Anderson, Vernon E. Anderson, Michael E. Harris & Eckhard Jankowsky

Nucleic-acid-binding proteins are generally viewed as either specific or nonspecific, depending on characteristics of their binding sites in DNA or RNA. Most studies have focused on specific proteins, which identify cognate sites by binding with highest affinities to regions with defined signatures in sequence, structure or both. Proteins that bind to sites devoid of defined sequence or structure signatures are considered nonspecific. Substrate binding by these proteins is poorly understood, and it is not known to what extent seemingly nonspecific proteins discriminate between different binding sites, aside from those sequestered by nucleic acid structures. Here we systematically examine substrate binding by the apparently nonspecific RNA-binding protein C5, and find clear discrimination between different binding site variants. C5 is the protein subunit of the transfer RNA processing ribonucleoprotein enzyme RNase P from Escherichia coli. The protein binds 5′ leaders of precursor tRNAs at a site without sequence or structure signatures. We measure functional binding of C5 to all possible sequence variants in its substrate binding site, using a high-throughput sequencing kinetics approach (HITS-KIN) that simultaneously follows processing of thousands of RNA species. C5 binds different substrate variants with affinities varying by orders of magnitude. The distribution of functional affinities of C5 for all substrate variants resembles affinity distributions of highly specific nucleic acid binding proteins. Unlike these specific proteins, C5 does not bind its physiological RNA targets with the highest affinity, but with affinities near the median of the distribution, a region that is not associated with a sequence signature. We delineate defined rules governing substrate recognition by C5, which reveal specificity that is hidden in cellular substrates for RNase P. Our findings suggest that apparently nonspecific and specific RNA-binding modes may not differ fundamentally, but represent distinct parts of common affinity distributions.

Nature Reviews Molecular Cell Biology 14, 612 (2013). doi:10.1038/nrm3673

Author: Rachel David

Staufen 1-mediated mRNA decay (SMD) is known to regulate the levels of transcripts involved in several cellular processes. SMD is triggered following binding of Staufen 1 to Staufen 1-binding sites (SBSs), which can form intramolecularly within an mRNA or through binding between an Alu element

Nature advance online publication 25 September 2013. doi:10.1038/nature12593

Authors: Takashi Nagano, Yaniv Lubling, Tim J. Stevens, Stefan Schoenfelder, Eitan Yaffe, Wendy Dean, Ernest D. Laue, Amos Tanay & Peter Fraser

Nature Methods 10, 925 (2013). doi:10.1038/nmeth.2630

Authors: Guy E Zinman, Shoshana Naiman, Yariv Kanfi, Haim Cohen & Ziv Bar-Joseph

Nature Methods 10, 921 (2013). doi:10.1038/nmeth.2659

Authors: Martin Krzywinski & Naomi Altman

The meaning of error bars is often misinterpreted, as is the statistical significance of their overlap.

Causes and Effects of N-Terminal Codon Bias in Bacterial Genes.

Science. 2013 Sep 26;

Authors: Goodman DB, Church GM, Kosuri S

Abstract
Most amino acids are encoded by multiple codons, and codon choice has strong effects on protein expression. Rare codons are enriched at the N terminus of genes in most organisms, although the causes and effects of this bias are unclear. Here, we measure expression from >14,000 synthetic reporters in Escherichia coli and show that using N-terminal rare codons instead of common ones increases expression by ~14-fold (median 4-fold). We quantify how individual N-terminal codons affect expression and show that these effects shape the sequence of natural genes. Finally, we demonstrate that reduced RNA structure and not codon rarity itself is responsible for expression increases. Our observations resolve controversies over the roles of N-terminal codon bias and suggest a straightforward method for optimizing heterologous gene expression in bacteria.

PMID: 24072823 [PubMed - as supplied by publisher]

Measurement of in vivo RNA synthesis rates.

Methods Enzymol. 2013;530:117-35

Authors: McPheeters DS, Wise JA

Abstract
A technique is described to directly measure ongoing transcription from individual genes in permeabilized cells of either the budding yeast Saccharomyces cerevisiae or the fission yeast Schizosaccharomyces pombe. Transcription run-on (TRO) analysis is used to compare the relative rates of synthesis for specific transcripts in cells grown under different environmental conditions or harvested at different stages of development. As the amount of an individual RNA species present at any given time is determined by its net rate of synthesis and degradation, an accurate picture of transcription per se can be obtained only by directly measuring de novo synthesis of RNA (if you are interested in RNA degradation, see Method for measuring mRNA decay rate in Saccharomyces cerevisiae). Most techniques employed to measure changes in the relative levels of individual transcripts present under different conditions, including Northern analysis (see Northern blotting), RT-PCR (see Reverse-transcription PCR (RT-PCR)), nuclease protection assays (see Explanatory Chapter: Nuclease Protection Assays), and genome-wide assays, such as microarray analysis and high throughput RNA sequencing, measure changes in the steady-state level of a transcript, which may or may not reflect the actual changes in transcription of the gene. Recent studies carried out in fission yeast have demonstrated that increases in the steady-state level (accumulation) of many individual mRNAs occur without any significant changes in transcription rates (McPheeters et al., 2009), highlighting the important role of regulated RNA stability in determining gene expression programs (Harigaya et al., 2006).

PMID: 24034318 [PubMed - in process]

Related Articles

The non-coding snRNA 7SK controls transcriptional termination, poising and bidirectionality in embryonic stem cells.

Genome Biol. 2013 Sep 17;14(9):r98

Authors: Castelo-Branco G, Amaral PP, Engström PG, Robson SC, Marques SC, Bertone P, Kouzarides T

Abstract
BACKGROUND: Pluripotency is characterized by a unique transcriptional state, in which lineage-specification genes are poised for transcription upon exposure to appropriate stimuli, via a bivalency mechanism involving the simultaneous presence of activating and repressive methylation marks at promoter-associated histones. Recent evidence suggests that other mechanisms, such as RNA polymerase II pausing, might be operational in this process, but their regulation remains poorly understood.
RESULTS: Here we identify the non-coding snRNA 7SK as a multifaceted regulator of transcription in embryonic stem cells. We find that 7SK represses a specific cohort of transcriptionally poised genes with bivalent or activating chromatin marks in these cells, suggesting a novel poising mechanism independent of Polycomb activity. Genome-wide analysis shows that 7SK also prevents transcription downstream of polyadenylation sites at several active genes, indicating that 7SK is required for normal transcriptional termination or control of 3[prime]-UTR length. In addition, 7SK suppresses divergent upstream antisense transcription at more than 2,600 loci, including many that encode divergent long non-coding RNAs, a finding that implicates the 7SK snRNA in the control of transcriptional bidirectionality.
CONCLUSIONS: Our study indicates that a single non-coding RNA, the snRNA 7SK, is a gatekeeper of transcriptional termination and bidirectional transcription in embryonic stem cells and mediates transcriptional poising through a mechanism independent of chromatin bivalency.

PMID: 24044525 [PubMed - as supplied by publisher]

Related Articles

Assessing gene-level translational control from ribosome profiling.

Bioinformatics. 2013 Sep 18;

Authors: Olshen AB, Hsieh AC, Stumpf CR, Olshen RA, Ruggero D, Taylor BS

Abstract
MOTIVATION: The translational landscape of diverse cellular systems remains largely uncharacterized. Indeed, a detailed understanding of the control of gene expression at the level of mRNA translation is vital to elucidating a systems-level view of complex molecular programs in the cell. Establishing the degree to which such post-transcriptional regulation can mediate specific phenotypes is similarly critical to elucidating the molecular pathogenesis of diseases such as cancer. Recently, methods for massively parallel sequencing of ribosome-bound fragments of messenger RNA have begun to uncover genome-wide translational control at codon-resolution. Despite its promise for deeply characterizing mammalian proteomes, few analytical methods exist for the comprehensive analysis of this paired RNA and ribosome data.
RESULTS: We describe the Babel framework, an analytical methodology for assessing the significance of changes in translational regulation within cells and between conditions. This approach facilitates the analysis of translation genome-wide while allowing statistically principled gene-level inference. Babel is based on an errors-in-variables regression model that utilizes the negative binomial distribution and draws inference using a parametric bootstrap approach. We demonstrate the operating characteristics of Babel on simulated data and use its gene-level inference to extend prior analyses significantly, discovering new translationally regulated modules under mTOR pathway signaling control.
AVAILABILITY: The Babel framework is freely available as source code at http://taylorlab.ucsf.edu/software_data.html.
CONTACT: barry.taylor@ucsf.edu SUPPLEMENTARY INFORMATION: Supplementary information is available at Bioinformatics online.

PMID: 24048356 [PubMed - as supplied by publisher]

It is a challenge to classify protein-coding or non-coding transcripts, especially those re-constructed from high-throughput sequencing data of poorly annotated species. This study developed and evaluated a powerful signature tool, Coding-Non-Coding Index (CNCI), by profiling adjoining nucleotide triplets to effectively distinguish protein-coding and non-coding sequences independent of known annotations. CNCI is effective for classifying incomplete transcripts and sense–antisense pairs. The implementation of CNCI offered highly accurate classification of transcripts assembled from whole-transcriptome sequencing data in a cross-species manner, that demonstrated gene evolutionary divergence between vertebrates, and invertebrates, or between plants, and provided a long non-coding RNA catalog of orangutan. CNCI software is available at http://www.bioinfo.org/software/cnci.

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.

GenBank is bursting with eukaryotic RNA sequencing (RNA-Seq) results. These data are transforming our view of nuclear transcriptional architecture, but many scientists are ignoring a major component of the data: mitochondrial- and chloroplast-derived sequences. Indeed, organelle transcripts typically represent a significant proportion of the reads generated from eukaryotic RNA-Seq experiments. Here, I argue that these data are an excellent and untapped resource for investigating many aspects of organelle function and evolution.

Summary: Unlike DNA, RNA abundances can vary over several orders of magnitude. Thus, identification of RNA–protein binding sites from high-throughput sequencing data presents unique challenges. Although peak identification in ChIP-Seq data has been extensively explored, there are few bioinformatics tools tailored for peak calling on analogous datasets for RNA-binding proteins. Here we describe ASPeak (abundance sensitive peak detection algorithm), an implementation of an algorithm that we previously applied to detect peaks in exon junction complex RNA immunoprecipitation in tandem experiments. Our peak detection algorithm yields stringent and robust target sets enabling sensitive motif finding and downstream functional analyses.

Availability: ASPeak is implemented in Perl as a complete pipeline that takes bedGraph files as input. ASPeak implementation is freely available at https://sourceforge.net/projects/as-peak under the GNU General Public License. ASPeak can be run on a personal computer, yet is designed to be easily parallelizable. ASPeak can also run on high performance computing clusters providing efficient speedup. The documentation and user manual can be obtained from http://master.dl.sourceforge.net/project/as-peak/manual.pdf.

Contact: alper.kucukural@umassmed.edu or ccenik@stanford.edu

by Saheli Chowdhury, Annemiek Dijkhuis, Sabrina Steiert, René Lutter

Interleukin 17A (IL-17), a mediator implicated in chronic and severe inflammatory diseases, enhances the production of pro-inflammatory mediators by attenuating decay of the encoding mRNAs. The decay of many of these mRNAs depends on proteins (AUBps) that target AU-rich elements in the 3′-untranslated region of mRNAs and facilitate either mRNA decay or stabilization. Here we show that AUBps and the target mRNA assemble in a novel ribonucleoprotein complex in the presence of microRNA16 (miR16), which leads to the degradation of the target mRNA. Notably, IL-17 attenuates miR16 expression and promotes the binding of stabilizing AUBps over that of destabilizing AUBps, reducing mRNA decay. These findings indicate that miR16 independently of a seed sequence, directs the competition between degrading and stabilizing AUBps for target mRNAs. Since AUBps affect expression of about 8% of the human transcriptome and miR16 is ubiquitously expressed, IL-17 may in addition to inflammation affect many other cellular processes.

Amanda N. Kallen, Xiao-Bo Zhou, Jie Xu, Chong Qiao, Jing Ma, Lei Yan, Lingeng Lu, Chaochun Liu, Jae-Sung Yi, Haifeng Zhang, Wang Min, Anton M. Bennett, Richard I. Gregory, Ye Ding, Yingqun Huang. Abundantly expressed in fetal tissues and adult muscle, the developmentally regulated H19 long noncoding RNA (lncRNA) has been implicated in human genetic disorders and cancer. However, how H19 ac....

Vihandha O. Wickramasinghe, Jane M. Savill, Sreenivas Chavali, Asta B. Jonsdottir, Eeson Rajendra, Tamara Grüner, Ronald A. Laskey, M. Madan Babu, Ashok R. Venkitaraman. Messenger RNA (mRNA) export from the nucleus is essential for eukaryotic gene expression. Here we identify a transcript-selective nuclear export mechanism affecting certain human transcripts, enri....

Daniel C. Koboldt, Karyn Meltz Steinberg, David E. Larson, Richard K. Wilson, Elaine R. Mardis. Genomics is a relatively new scientific discipline, having DNA sequencing as its core technology. As technology has improved the cost and scale of genome characterization over sequencing’s 40-year....