Hyeshik Chang

Cytoplasmic intron retention, function, splicing, and the sentinel RNA hypothesis.

Wiley Interdiscip Rev RNA. 2013 Nov 4;

Authors: Buckley PT, Khaladkar M, Kim J, Eberwine J

Abstract
Cytoplasmic splicing represents a newly emerging level of transcriptional regulation adding to the molecular diversity of mammalian cells. As examples of this noncanonical form of transcript processing are discovered, the evidence of its importance to normal cellular function grows. Work from a number of groups using a variety of cell types is steadily identifying a large number of transcripts (and soon to be even larger as genome-wide analyses of retained introns across a number of cellular phenotypes are currently underway) that undergo some level of regulated endogenous extranuclear splicing as part of their normal biosynthetic pathway. Here, we review the existing data covering cytoplasmic retained intron sequences and suggest that such sequences may be a component of 'sentinel RNA' that serves to generate transcript variants within the cytoplasm as well as a source for RNA-based secondary messages. For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.

PMID: 24190870 [PubMed - as supplied by publisher]

Background: Dynamics of polyadenylation vs. deadenylation determine the fate of several developmentally regulated genes. Decay of a subset of maternal mRNAs and new transcription define the maternal-to-zygotic transition, but the full complement of polyadenylated and deadenylated coding and non-coding transcripts has not yet been assessed in Xenopus embryos Results: To analyze the dynamics and diversity of coding and non-coding transcripts during development, both polyadenylated mRNA and ribosomal RNA-depleted total RNA were harvested across six developmental stages and subjected to high throughput sequencing. The maternally loaded transcriptome is highly diverse and consists of both polyadenylated and deadenylated transcripts. Many maternal genes show peak expression in the oocyte and include genes which are known to be the key regulators of events like oocyte maturation and fertilization. Of all the transcripts that increase in abundance between early blastula and larval stages, about 30\% of the embryonic genes are induced by fourfold or more by the late blastula stage and another 35\% by late gastrulation. Using a gene model validation and discovery pipeline, we identified novel transcripts and putative long non-coding RNAs (lncRNA). These lncRNA transcripts were stringently selected as spliced transcripts generated from independent promoters, with limited coding potential and a codon bias characteristic of noncoding sequences. Many lncRNAs are conserved and expressed in a developmental stage-specific fashion. Conclusions: These data reveal dynamics of transcriptome polyadenylation and abundance and provides a high-confidence catalogue of novel and long non-coding RNAs.

Nature advance online publication 06 November 2013. doi:10.1038/nature12734

Authors: Sebastian M. Fica, Nicole Tuttle, Thaddeus Novak, Nan-Sheng Li, Jun Lu, Prakash Koodathingal, Qing Dai, Jonathan P. Staley & Joseph A. Piccirilli

A multifunctional protein EWS regulates the expression of Drosha and microRNAs.

Cell Death Differ. 2013 Nov 1;

Authors: Kim KY, Hwang YJ, Jung MK, Choe J, Kim Y, Kim S, Lee CJ, Ahn H, Lee J, Kowall NW, Kim YK, Kim JI, Lee SB, Ryu H

Abstract
EWS (Ewing's Sarcoma) gene encodes an RNA/DNA-binding protein that is ubiquitously expressed and involved in various cellular processes. EWS deficiency leads to impaired development and early senescence through unknown mechanisms. We found that EWS regulates the expression of Drosha and microRNAs (miRNAs). EWS deficiency resulted in increased expression of Drosha, a well-known microprocessor, and increased levels of miR-29b and miR-18b. Importantly, miR-29b and miR-18b were directly involved in the post-transcriptional regulation of collagen IV alpha 1 (Col4a1) and connective tissue growth factor (CTGF) in EWS knock-out (KO) mouse embryonic fibroblast cells. The upregulation of Drosha, miR-29b and miR-18b and the sequential downregulation of Col4a1 and CTGF contributed to the deregulation of dermal development in EWS KO mice. Otherwise, knockdown of Drosha rescued miRNA-dependent downregulation of Col4a1 and CTGF proteins. Taken together, our data indicate that EWS is involved in post-transcriptional regulation of Col4a1 and CTGF via a Drosha-miRNA-dependent pathway. This finding suggests that EWS has a novel role in dermal morphogenesis through the modulation of miRNA biogenesis.Cell Death and Differentiation advance online publication, 1 November 2013; doi:10.1038/cdd.2013.144.

PMID: 24185621 [PubMed - as supplied by publisher]

Related Articles

Evolutionary conservation of long noncoding RNAs; sequence, structure, function.

Biochim Biophys Acta. 2013 Oct 26;

Authors: Johnsson P, Lipovich L, Grandér D, Morris KV

Abstract
BACKGROUND:: Recent advances in genome wide studies have revealed the abundance of long non-coding RNAs (lncRNAs) in mammalian transcriptomes. The ENCODE Consortium has elucidated the prevalence of human lncRNA genes, which are as numerous as protein-coding genes. Surprisingly, many lncRNAs do not show the same pattern of high interspecies conservation as protein-coding genes. The absence of functional studies and the frequent lack of sequence conservation therefore make functional interpretation of these newly discovered transcripts challenging. Many investigators have suggested the presence and importance of secondary structural elements within lncRNAs, but mammalian lncRNA secondary structure remains poorly understood. It is intriguing to speculate that in this group of genes, RNA secondary structures might be preserved throughout evolution and that this might explain the lack of sequence conservation among many lncRNAs. Scope of review: Here, we review the extent of interspecies conservation among different lncRNAs, with a focus on a subset of lncRNAs that have been functionally investigated. The function of lncRNAs is widespread and we investigate whether different forms of functionalities may be conserved. Major conclusions: Lack of conservation does not imbue a lack of function. We highlight several examples of lncRNAs where RNA structure appears to be the main functional unit and evolutionary constraint. We survey existing genomewide studies of mammalian lncRNA conservation and summarize their limitations. We further review specific human lncRNAs which lack evolutionary conservation beyond primates but have proven to be both functional and therapeutically relevant. General significance: Pioneering studies highlight a role in lncRNAs for secondary structures, and possibly the presence of functional "modules", which are interspersed with longer and less conserved stretches of nucleotide sequences. Taken together, high-throughput analysis of conservation and functional composition of the still-mysterious lncRNA genes is only now becoming feasible.

PMID: 24184936 [PubMed - as supplied by publisher]

Related Articles

GWIPS-viz: development of a ribo-seq genome browser.

Nucleic Acids Res. 2013 Oct 31;

Authors: Michel AM, Fox G, M Kiran A, De Bo C, O'Connor PB, Heaphy SM, Mullan JP, Donohue CA, Higgins DG, Baranov PV

Abstract
We describe the development of GWIPS-viz (http://gwips.ucc.ie), an online genome browser for viewing ribosome profiling data. Ribosome profiling (ribo-seq) is a recently developed technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome-protected messenger RNA (mRNA) fragments, which allows the ribosome density along all mRNA transcripts present in the cell to be quantified. Since its inception, ribo-seq has been carried out in a number of eukaryotic and prokaryotic organisms. Owing to the increasing interest in ribo-seq, there is a pertinent demand for a dedicated ribo-seq genome browser. GWIPS-viz is based on The University of California Santa Cruz (UCSC) Genome Browser. Ribo-seq tracks, coupled with mRNA-seq tracks, are currently available for several genomes: human, mouse, zebrafish, nematode, yeast, bacteria (Escherichia coli K12, Bacillus subtilis), human cytomegalovirus and bacteriophage lambda. Our objective is to continue incorporating published ribo-seq data sets so that the wider community can readily view ribosome profiling information from multiple studies without the need to carry out computational processing.

PMID: 24185699 [PubMed - as supplied by publisher]

Related Articles

iCLIP: Protein-RNA interactions at nucleotide resolution.

Methods. 2013 Oct 24;

Authors: Huppertz I, Attig J, D'Ambrogio A, Eastonb L, Sibley CR, Sugimoto Y, Tajnik M, König J, Ule J

Abstract
RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.

PMID: 24184352 [PubMed - as supplied by publisher]

Related Articles

Systematic evaluation of spliced alignment programs for RNA-seq data.

Nat Methods. 2013 Nov 3;

Authors: Engström PG, Steijger T, Sipos B, Grant GR, Kahles A, The RGASP Consortium, Alioto T, Behr J, Bertone P, Bohnert R, Campagna D, Davis CA, Dobin A, Engström PG, Gingeras TR, Goldman N, Grant GR, Guigó R, Harrow J, Hubbard TJ, Jean G, Kahles A, Kosarev P, Li S, Liu J, Mason CE, Molodtsov V, Ning Z, Ponstingl H, Prins JF, Rätsch G, Ribeca P, Seledtsov I, Sipos B, Solovyev V, Steijger T, Valle G, Vitulo N, Wang K, Wu TD, Zeller G, Rätsch G, Goldman N, Hubbard TJ, Harrow J, Guigó R, Bertone P

Abstract
High-throughput RNA sequencing is an increasingly accessible method for studying gene structure and activity on a genome-wide scale. A critical step in RNA-seq data analysis is the alignment of partial transcript reads to a reference genome sequence. To assess the performance of current mapping software, we invited developers of RNA-seq aligners to process four large human and mouse RNA-seq data sets. In total, we compared 26 mapping protocols based on 11 programs and pipelines and found major performance differences between methods on numerous benchmarks, including alignment yield, basewise accuracy, mismatch and gap placement, exon junction discovery and suitability of alignments for transcript reconstruction. We observed concordant results on real and simulated RNA-seq data, confirming the relevance of the metrics employed. Future developments in RNA-seq alignment methods would benefit from improved placement of multimapped reads, balanced utilization of existing gene annotation and a reduced false discovery rate for splice junctions.

PMID: 24185836 [PubMed - as supplied by publisher]

Related Articles

Assessment of transcript reconstruction methods for RNA-seq.

Nat Methods. 2013 Nov 3;

Authors: Steijger T, Abril JF, Engström PG, Kokocinski F, The RGASP Consortium, Abril JF, Akerman M, Alioto T, Ambrosini G, Antonarakis SE, Behr J, Bertone P, Bohnert R, Bucher P, Cloonan N, Derrien T, Djebali S, Du J, Dudoit S, Engström PG, Gerstein M, Gingeras TR, Gonzalez D, Grimmond SM, Guigó R, Habegger L, Harrow J, Hubbard TJ, Iseli C, Jean G, Kahles A, Kokocinski F, Lagarde J, Leng J, Lefebvre G, Lewis S, Mortazavi A, Niermann P, Rätsch G, Reymond A, Ribeca P, Richard H, Rougemont J, Rozowsky J, Sammeth M, Sboner A, Schulz MH, Searle SM, Solorzano ND, Solovyev V, Stanke M, Steijger T, Stevenson BJ, Stockinger H, Valsesia A, Weese D, White S, Wold BJ, Wu J, Wu TD, Zeller G, Zerbino D, Zhang MQ, Hubbard TJ, Guigó R, Harrow J, Bertone P

Abstract
We evaluated 25 protocol variants of 14 independent computational methods for exon identification, transcript reconstruction and expression-level quantification from RNA-seq data. Our results show that most algorithms are able to identify discrete transcript components with high success rates but that assembly of complete isoform structures poses a major challenge even when all constituent elements are identified. Expression-level estimates also varied widely across methods, even when based on similar transcript models. Consequently, the complexity of higher eukaryotic genomes imposes severe limitations on transcript recall and splice product discrimination that are likely to remain limiting factors for the analysis of current-generation RNA-seq data.

PMID: 24185837 [PubMed - as supplied by publisher]

miR-17∼92 is a polycistronic microRNA (miR) cluster (consisting of miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a) which frequently is overexpressed in several solid and lymphoid malignancies. Loss- and gain-of-function studies have revealed the role of miR-17∼92 in heart, lung, and B-cell development and in Myc-induced B-cell lymphomas, respectively. Recent studies...

Adenosine deaminases that act on RNA are a conserved family of enzymes that catalyze a natural process of site-directed mutagenesis. Biochemically, they convert adenosine to inosine, a nucleotide that is read as guanosine during translation; thus when editing occurs in mRNAs, codons can be recoded and the changes can alter...

by Yanmei Huang, Joshua A. Ainsley, Leon G. Reijmers, F. Rob Jackson

Abstract

Genome-wide studies of circadian transcription or mRNA translation have been hindered by the presence of heterogeneous cell populations in complex tissues such as the nervous system. We describe here the use of a Drosophila cell-specific translational profiling approach to document the rhythmic “translatome” of neural clock cells for the first time in any organism. Unexpectedly, translation of most clock-regulated transcripts—as assayed by mRNA ribosome association—occurs at one of two predominant circadian phases, midday or mid-night, times of behavioral quiescence; mRNAs encoding similar cellular functions are translated at the same time of day. Our analysis also indicates that fundamental cellular processes—metabolism, energy production, redox state (e.g., the thioredoxin system), cell growth, signaling and others—are rhythmically modulated within clock cells via synchronized protein synthesis. Our approach is validated by the identification of mRNAs known to exhibit circadian changes in abundance and the discovery of hundreds of novel mRNAs that show translational rhythms. This includes Tdc2, encoding a neurotransmitter synthetic enzyme, which we demonstrate is required within clock neurons for normal circadian locomotor activity.

Cell-lineage–specific transcripts are essential for differentiated tissue function, implicated in hereditary organ failure, and mediate acquired chronic diseases. However, experimental identification of cell-lineage–specific genes in a genome-scale manner is infeasible for most solid human tissues. We developed the first genome-scale method to identify genes with cell-lineage–specific expression, even in lineages not separable by experimental microdissection. Our machine-learning–based approach leverages high-throughput data from tissue homogenates in a novel iterative statistical framework. We applied this method to chronic kidney disease and identified transcripts specific to podocytes, key cells in the glomerular filter responsible for hereditary and most acquired glomerular kidney disease. In a systematic evaluation of our predictions by immunohistochemistry, our in silico approach was significantly more accurate (65% accuracy in human) than predictions based on direct measurement of in vivo fluorescence-tagged murine podocytes (23%). Our method identified genes implicated as causal in hereditary glomerular disease and involved in molecular pathways of acquired and chronic renal diseases. Furthermore, based on expression analysis of human kidney disease biopsies, we demonstrated that expression of the podocyte genes identified by our approach is significantly related to the degree of renal impairment in patients. Our approach is broadly applicable to define lineage specificity in both cell physiology and human disease contexts. We provide a user-friendly website that enables researchers to apply this method to any cell-lineage or tissue of interest. Identified cell-lineage–specific transcripts are expected to play essential tissue-specific roles in organogenesis and disease and can provide starting points for the development of organ-specific diagnostics and therapies.

The Gene Promoter Expression Prediction challenge consisted of predicting gene expression from promoter sequences in a previously unknown experimentally generated data set. The challenge was presented to the community in the framework of the sixth Dialogue for Reverse Engineering Assessments and Methods (DREAM6), a community effort to evaluate the status of systems biology modeling methodologies. Nucleotide-specific promoter activity was obtained by measuring fluorescence from promoter sequences fused upstream of a gene for yellow fluorescence protein and inserted in the same genomic site of yeast Saccharomyces cerevisiae. Twenty-one teams submitted results predicting the expression levels of 53 different promoters from yeast ribosomal protein genes. Analysis of participant predictions shows that accurate values for low-expressed and mutated promoters were difficult to obtain, although in the latter case, only when the mutation induced a large change in promoter activity compared to the wild-type sequence. As in previous DREAM challenges, we found that aggregation of participant predictions provided robust results, but did not fare better than the three best algorithms. Finally, this study not only provides a benchmark for the assessment of methods predicting activity of a specific set of promoters from their sequence, but it also shows that the top performing algorithm, which used machine-learning approaches, can be improved by the addition of biological features such as transcription factor binding sites.

RNA transcripts are generally identical to the underlying DNA sequences. Nevertheless, RNA–DNA differences (RDDs) were found in the nuclear human genome and in plants and animals but not in human mitochondria. Here, by deep sequencing of human mitochondrial DNA (mtDNA) and RNA, we identified three RDD sites at mtDNA positions 295 (C-to-U), 13710 (A-to-U, A-to-G), and 2617 (A-to-U, A-to-G). Position 2617, within the 16S rRNA, harbored the most prevalent RDDs (>30% A-to-U and ~15% A-to-G of the reads in all tested samples). The 2617 RDDs appeared already at the precursor polycistrone mitochondrial transcript. By using traditional Sanger sequencing, we identified the A-to-U RDD in six different cell lines and representative primates (Gorilla gorilla, Pongo pigmaeus, and Macaca mulatta), suggesting conservation of the mechanism generating such RDD. Phylogenetic analysis of more than 1700 vertebrate mtDNA sequences supported a thymine as the primate ancestral allele at position 2617, suggesting that the 2617 RDD recapitulates the ancestral 16S rRNA. Modeling U or G (the RDDs) at position 2617 stabilized the large ribosomal subunit structure in contrast to destabilization by an A (the pre-RDDs). Hence, these mitochondrial RDDs are likely functional.

The lack of long-term evolutionary conservation of microRNA (miRNA) target sites appears to contradict many analyses of their functions. Several hypotheses have been offered, but an attractive one—that the conservation may be a function of taxonomic hierarchy (vertebrates, mammals, primates, etc.)—has rarely been discussed. For such an analysis, we cannot use evolutionary conservation as a criterion of target identification, and hence, have used high confidence target sites in the cross-linking immunoprecipitation (CLIP) data. Assuming that a proportion, p, of target sites in the CLIP data are conserved, we define the evolvability of miRNA targets as 1-p. Genomic data from vertebrate species show that the evolvability between human and fish is very high, at more than 90%. The evolvability decreases to 50% between birds and mammals, 20% among mammalian orders, and only 6% between human and chimpanzee. Within each taxonomic hierarchy, there is a set of targets that are conserved only at that level of evolution. Extrapolating the evolutionary trend, we find the evolvability in any single species to be close to 0%. Thus, all miRNA target sites identified by the CLIP method are evolutionarily conserved in one species, but the conservation is lost step by step in larger taxonomic groups. The changing evolvability of miRNA targets suggests that miRNA-target interactions may play a role in the evolution of organismal diversity.

Information about the secondary structure of RNA is often useful when assessing the potential for certain RNAs to interact with proteins or when determining whether RNAs that are dissimilar in sequence can form the same structure. In this protocol we discuss chemical methods for RNA structure determination. These methods rely on the fact that certain reagents interact with RNA bases when they are single stranded, but do not react when the bases are involved in Watson–Crick base pairs. For example, dimethylsulfate (DMS) methylates the N1 position of adenosine, the N7 position of guanine, and the N3 position of cytosine only when these bases are in single-strand regions. Modifications of adenosine and cytosine create blocks to reverse transcriptase; accordingly, these modifications are detected as stops to primer extension. Modification of guanine does not create reverse transcriptase stops, but these modifications can be detected by cleavage of the modified RNA after borohydride reduction and aniline cleavage. Because DMS and other chemical reagents modify only single-stranded RNA, double-stranded regions are inferred by the lack of modification.

Despite studies that have investigated the interactions of double-stranded RNA-binding proteins like Staufen with RNA in vitro, how they achieve target specificity in vivo remains uncertain. We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos. Analysis of the localization and functions of these transcripts revealed a number of potentially novel roles for Staufen. Using computational methods, we identified two sequence features that distinguish Staufen’s target transcripts from non-targets. First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3–4-fold longer than unbound transcripts. Second, the 3'UTRs of Staufen-bound transcripts are highly enriched for three types of secondary structures. These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs. Our results provide the first systematic genome-wide analysis showing how a double-stranded RNA-binding protein achieves target specificity.

Use of transcription activator-like effector nucleases (TALENs) is a promising new technique in the field of targeted genome engineering, editing and reverse genetics. Its applications span from introducing knockout mutations to endogenous tagging of proteins and targeted excision repair. Owing to this wide range of possible applications, there is a need for fast and user-friendly TALEN design tools. We developed E-TALEN (http://www.e-talen.org), a web-based tool to design TALENs for experiments of varying scale. E-TALEN enables the design of TALENs against a single target or a large number of target genes. We significantly extended previously published design concepts to consider genomic context and different applications. E-TALEN guides the user through an end-to-end design process of de novo TALEN pairs, which are specific to a certain sequence or genomic locus. Furthermore, E-TALEN offers a functionality to predict targeting and specificity for existing TALENs. Owing to the computational complexity of many of the steps in the design of TALENs, particular emphasis has been put on the implementation of fast yet accurate algorithms. We implemented a user-friendly interface, from the input parameters to the presentation of results. An additional feature of E-TALEN is the in-built sequence and annotation database available for many organisms, including human, mouse, zebrafish, Drosophila and Arabidopsis, which can be extended in the future.

Nature Reviews Drug Discovery 12, 847 (2013). doi:10.1038/nrd4140

Authors: Hui Ling, Muller Fabbri & George A. Calin

The first cancer-targeted microRNA (miRNA) drug — MRX34, a liposome-based miR-34 mimic — entered Phase I clinical trials in patients with advanced hepatocellular carcinoma in April 2013, and miRNA therapeutics are attracting special attention from both academia and biotechnology companies. Although miRNAs are the most

Chris Gunter on an editor's experience of scientific conferences - and how researchers can make the most of these encounters

by Virginie Bernard, Magali Michaut

What are you working on? You have certainly been asked that question many times, whether it be at a Saturday night party, during a discussion with your neighbors, or at a family gathering. Communicating with a lay audience about scientific subjects and making them attractive is a difficult task. But difficult or not, you will have to do it for many years, not only with your family and friends, but also with your colleagues and collaborators. So, better learn now! Although not usually taught, the ability to explain your work to others is an essential skill in science, where communication plays a key role. Using some examples of the French Regional Student Group activities, we discuss here (i) why it is important to have such communication skills, (ii) how you can get involved in these activities by using existing resources or working with people who have previous experience, and (iii) what you get out of this amazing experience. We aim to motivate you and provide you with tips and ideas to get involved in promoting scientific activities while getting all the benefits.

In Vivo Processing Assay Based on a Dual-Luciferase Reporter System to Evaluate DROSHA Enzymatic Activity.

Methods Mol Biol. 2014;1095:87-93

Authors: Bilan V, Allegra D, Kuchenbauer F, Mertens D

Abstract
Luciferase reporter assays are widely used to study promoter activity, transcription factors, intracellular signaling, protein interactions (Jia et al., PloS One 6:e26414), miRNA processing (Allegra and Mertens, Biochem Biophys Res Commun 406:501-505), and target recognition (Jin et al., Methods Mol Biol 936:117-127). Here we describe the use of a dual-luciferase reporter system to evaluate the enzymatic activity of a key enzyme involved in RNA maturation-DROSHA. This dual system is a simple and fast method for the quantification of the DROSHA processing activity in live cells.

PMID: 24166304 [PubMed - in process]

Nature Methods 10, 1056 (2013). doi:10.1038/nmeth.2705

Crystal structure of the 14-subunit RNA polymerase I

Nature 502, 7473 (2013). doi:10.1038/nature12636

Authors: Carlos Fernández-Tornero, María Moreno-Morcillo, Umar J. Rashid, Nicholas M. I. Taylor, Federico M. Ruiz, Tim Gruene, Pierre Legrand, Ulrich Steuerwald & Christoph W. Müller

Protein biosynthesis depends on the availability of ribosomes, which in turn relies on ribosomal RNA production. In eukaryotes, this process is carried out by RNA polymerase I (Pol I), a 14-subunit enzyme, the activity of which is a major determinant of cell growth. Here we

RNA polymerase I structure and transcription regulation

Nature 502, 7473 (2013). doi:10.1038/nature12712

Authors: Christoph Engel, Sarah Sainsbury, Alan C. Cheung, Dirk Kostrewa & Patrick Cramer

Transcription of ribosomal RNA by RNA polymerase (Pol) I initiates ribosome biogenesis and regulates eukaryotic cell growth. The crystal structure of Pol I from the yeast Saccharomyces cerevisiae at 2.8 Å resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II.

Nature Genetics 45, 1337 (2013). doi:10.1038/ng.2763

Authors: Dong hyun Kim, Dominic Grün & Alexander van Oudenaarden

Summary: In RNA design problems, it is plausible to assume that the user would be interested in preserving a particular RNA secondary structure motif, or fragment, for biological reasons. The preservation could be in structure or sequence, or both. Thus, the inverse RNA folding problem could benefit from considering fragment constraints.

We have developed a new interactive Java application called RNA fragment-based inverse that allows users to insert an RNA secondary structure in dot-bracket notation. It then performs sequence design that conforms to the shape of the input secondary structure, the specified thermodynamic stability, the specified mutational robustness and the user-selected fragment after shape decomposition. In this shape-based design approach, specific RNA structural motifs with known biological functions are strictly enforced, while others can possess more flexibility in their structure in favor of preserving physical attributes and additional constraints.

Availability: RNAfbinv is freely available for download on the web at http://www.cs.bgu.ac.il/~RNAexinv/RNAfbinv. The site contains a help file with an explanation regarding the exact use.

Contact: dbarash@cs.bgu.ac.il

Summary: Here we introduce catRAPID omics, a server for large-scale calculations of protein–RNA interactions. Our web server allows (i) predictions at proteomic and transcriptomic level; (ii) use of protein and RNA sequences without size restriction; (iii) analysis of nucleic acid binding regions in proteins; and (iv) detection of RNA motifs involved in protein recognition.

Results: We developed a web server to allow fast calculation of ribonucleoprotein associations in Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, Homo sapiens, Mus musculus, Rattus norvegicus, Saccharomyces cerevisiae and Xenopus tropicalis (custom libraries can be also generated). The catRAPID omics was benchmarked on the recently published RNA interactomes of Serine/arginine-rich splicing factor 1 (SRSF1), Histone-lysine N-methyltransferase EZH2 (EZH2), TAR DNA-binding protein 43 (TDP43) and RNA-binding protein FUS (FUS) as well as on the protein interactomes of U1/U2 small nucleolar RNAs, X inactive specific transcript (Xist) repeat A region (RepA) and Crumbs homolog 3 (CRB3) 3'-untranslated region RNAs. Our predictions are highly significant (P < 0.05) and will help the experimentalist to identify candidates for further validation.

Availability: catRAPID omics can be freely accessed on the Web at http://s.tartaglialab.com/catrapid/omics. Documentation, tutorial and FAQs are available at http://s.tartaglialab.com/page/catrapid_group.

Contact: gian.tartaglia@crg.eu

Nature advance online publication 27 October 2013. doi:10.1038/nature12719

Authors: Matthew D. Simon, Stefan F. Pinter, Rui Fang, Kavitha Sarma, Michael Rutenberg-Schoenberg, Sarah K. Bowman, Barry A. Kesner, Verena K. Maier, Robert E. Kingston & Jeannie T. Lee

The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes. During XCI, Xist coats the future inactive X chromosome (Xi) and recruits Polycomb repressive complex 2 (PRC2) to the X-inactivation centre (Xic). How Xist spreads silencing on a 150-megabases scale is unclear. Here we generate high-resolution maps of Xist binding on the X chromosome across a developmental time course using CHART-seq. In female cells undergoing XCI de novo, Xist follows a two-step mechanism, initially targeting gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but may concentrate near escapee boundaries. Xist binding is linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), indicating co-migration of Xist and PRC2. Interestingly, when Xist is acutely stripped off from the Xi in post-XCI cells, Xist recovers quickly within both gene-rich and gene-poor domains on a timescale of hours instead of days, indicating a previously primed Xi chromatin state. We conclude that Xist spreading takes distinct stage-specific forms. During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and gene-poor regions.