Hyeshik Chang

RNA-seq is a methodology for RNA profiling based on next-generation sequencing that enables to measure and compare gene expression patterns at unprecedented resolution. Although the appealing features of this technique have promoted its application to a wide panel of transcriptomics studies, the fast-evolving nature of experimental protocols and computational tools challenges the definition of a unified RNA-seq analysis pipeline. In this review, focused on the study of differential gene expression with RNA-seq, we go through the main steps of data processing and discuss open challenges and possible solutions.

Formaldehyde cross-linking is an important component of many technologies, including chromatin immunoprecipitation and chromosome conformation capture. The procedure remains empirical and poorly characterized, however, despite a long history of its use in research. Little is known about the specificity of in vivo cross-linking, its efficiency and chemical adducts induced by the procedure. It is time to search this black box.

by Aymeric Chartier, Pierre Klein, Stéphanie Pierson, Nicolas Barbezier, Teresa Gidaro, François Casas, Steven Carberry, Paul Dowling, Laurie Maynadier, Maëlle Bellec, Martine Oloko, Claude Jardel, Bodo Moritz, George Dickson, Vincent Mouly, Kay Ohlendieck, Gillian Butler-Browne, Capucine Trollet, Martine Simonelig

Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.

Article

The identification of factors involved in eukaryotic DNA regulation at specific genomic regions distinct technical challenges. Here, the authors describe ePICh, a method that allows for the efficient isolation of chromatin factors associated with complex low abundance targets within the large genome of mammalian cells.

Nature Communications doi: 10.1038/ncomms7674

Authors: Satoru Ide, Jerome Dejardin

Publication date: May 2015
Source:Trends in Biochemical Sciences, Volume 40, Issue 5
Author(s): Sonia Guil, Manel Esteller
The past few years have witnessed an exciting increase in the richness and complexity of RNA-mediated regulatory circuitries, including new types of RNA–RNA interaction that underlie key steps in gene expression control in an organized and probably hierarchic system to dictate final protein output. Both small (especially miRNAs) and long coding (lc) and noncoding (nc) RNAs contain structural domains that can sense and bind other RNAs via complementary base pairing. The versatility of the interaction confers multiple roles to RNA–RNA hybrids, from control of RNA biogenesis to competition for common targets. Here, we focus on the emerging evidence around RNA networks and their impact on gene expression regulation in light of recent breakthroughs around the crosstalk between coding RNAs and ncRNAs.

Publication date: 31 March 2015
Source:Cell Reports, Volume 10, Issue 12
Author(s): Elke F. Roovers , David Rosenkranz , Mahdi Mahdipour , Chung-Ting Han , Nannan He , Susana M. Chuva de Sousa Lopes , Lucette A.J. van der Westerlaken , Hans Zischler , Falk Butter , Bernard A.J. Roelen , René F. Ketting
Germ cells of most animals critically depend on piRNAs and Piwi proteins. Surprisingly, piRNAs in mouse oocytes are relatively rare and dispensable. We present compelling evidence for strong Piwi and piRNA expression in oocytes of other mammals. Human fetal oocytes express PIWIL2 and transposon-enriched piRNAs. Oocytes in adult human ovary express PIWIL1 and PIWIL2, whereas those in bovine ovary only express PIWIL1. In human, macaque, and bovine ovaries, we find piRNAs that resemble testis-borne pachytene piRNAs. Isolated bovine follicular oocytes were shown to contain abundant, relatively short piRNAs that preferentially target transposable elements. Using label-free quantitative proteome analysis, we show that these maturing oocytes strongly and specifically express the PIWIL3 protein, alongside other, known piRNA-pathway components. A piRNA pool is still present in early bovine embryos, revealing a potential impact of piRNAs on mammalian embryogenesis. Our results reveal that there are highly dynamic piRNA pathways in mammalian oocytes and early embryos.

Graphical abstract

Teaser

Piwi proteins and piRNAs are essential for animal germ cells, yet their presence in mammalian oocytes has remained enigmatic. Using bovine, macaque, and human material, Roovers et al. demonstrate dynamic expression of these molecules during oogenesis. Notably, PIWIL3 is found specifically in oocytes, together with a non-methylated, adenylated piRNA population.

Altered microRNA production in cancer promotes cell proliferation, migration, survival, and adaptation to new environments.

Related Articles

Competition between target sites of regulators shapes post-transcriptional gene regulation.

Nat Rev Genet. 2015 Feb;16(2):113-26

Authors: Jens M, Rajewsky N

Abstract
Post-transcriptional gene regulation (PTGR) of mRNA turnover, localization and translation is mediated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). These regulators exert their effects by binding to specific sequences within their target mRNAs. Increasing evidence suggests that competition for binding is a fundamental principle of PTGR. Not only can miRNAs be sequestered and neutralized by the targets with which they interact through a process termed 'sponging', but competition between binding sites on different RNAs may also lead to regulatory crosstalk between transcripts. Here, we quantitatively model competition effects under physiological conditions and review the role of endogenous sponges for PTGR in light of the key features that emerge.

PMID: 25488579 [PubMed - indexed for MEDLINE]

Analysis of RNA decay factor mediated RNA stability contributions on RNA abundance.

BMC Genomics. 2015 Dec;16(1):1358

Authors: Maekawa S, Imamachi N, Irie T, Tani H, Matsumoto K, Mizutani R, Imamura K, Kakeda M, Yada T, Sugano S, Suzuki Y, Akimitsu N

Abstract
BACKGROUND: Histone epigenome data determined by chromatin immunoprecipitation sequencing (ChIP-seq) is used in identifying transcript regions and estimating expression levels. However, this estimation does not always correlate with eventual RNA expression levels measured by RNA sequencing (RNA-seq). Part of the inconsistency may arise from the variance in RNA stability, where the transcripts that are more or less abundant than predicted RNA expression from histone epigenome data are inferred to be more or less stable. However, there is little systematic analysis to validate this assumption. Here, we used stability data of whole transcriptome measured by 5'-bromouridine immunoprecipitation chase sequencing (BRIC-seq), which enabled us to determine the half-lives of whole transcripts including lincRNAs, and we integrated BRIC-seq with ChIP-seq to achieve better estimation of the eventual transcript levels and to understand the importance of post-transcriptional regulation that determine the eventual transcript levels.
RESULTS: We identified discrepancies between the RNA abundance estimated by ChIP-seq and measured RNA expression from RNA-seq; for number of genes and estimated that the expression level of 865 genes was controlled at the level of RNA stability in HeLa cells. ENCODE data analysis supported the idea that RNA stability control aids to determine transcript levels in multiple cell types. We identified UPF1, EXOSC5 and STAU1, well-studied RNA degradation factors, as controlling factors for 8% of cases. Computational simulations reasonably explained the changes of eventual mRNA levels attributable to the changes in the rates of mRNA half-lives. In addition, we propose a feedback circuit that includes the regulated degradation of mRNAs encoding transcription factors to maintain the steady state level of RNA abundance. Intriguingly, these regulatory mechanisms were distinct between mRNAs and lincRNAs.
CONCLUSIONS: Integrative analysis of ChIP-seq, RNA-seq and our BRIC-seq showed that transcriptional regulation and RNA degradation are independently regulated. In addition, RNA stability is an important determinant of eventual transcript levels. RNA binding proteins, such as UPF1, STAU1 and EXOSC5 may play active roles in such controls.

PMID: 25776694 [PubMed - in process]

Polyadenylation [poly(A)] is a vital step in post-transcriptional processing of pre-mRNA. Alternative polyadenylation is a widespread mechanism of regulating gene expression in eukaryotes. Defining poly(A) sites contributes to the annotation of transcripts’ ends and the study of gene regulatory mechanisms. Here, we survey methods for collecting poly(A) sites using high-throughput sequencing technologies and summarize the general processes for genome-wide poly(A) site identifications. We also compare the performances of various poly(A) site prediction models and discuss the relationship between poly(A) site identification from sequencing projects and predictive modeling. Moreover, we attempt to address some potential problems in current researches and propose future directions related to polyadenylation research.

Nature Chemical Biology 11, 241 (2015). doi:10.1038/nchembio.1782

Author: Stephen Davey

Article

As important post-transcriptional regulators of gene expression, microRNAs play a key role in the generation of complex phenotypes. Here, Huan et al. identify miR-eQTLs in whole blood samples to create a roadmap linking regulation of microRNA expression to complex diseases.

Nature Communications doi: 10.1038/ncomms7601

Authors: Tianxiao Huan, Jian Rong, Chunyu Liu, Xiaoling Zhang, Kahraman Tanriverdi, Roby Joehanes, Brian H. Chen, Joanne M. Murabito, Chen Yao, Paul Courchesne, Peter J. Munson, Christopher J. O’Donnell, Nancy Cox, Andrew D. Johnson, Martin G. Larson, Daniel Levy, Jane E. Freedman

miR-RACE: An Effective Approach to Accurately Determine the Sequence of Computationally Identified miRNAs.

Methods Mol Biol. 2015;1296:109-18

Authors: Wang C, Fang J

Abstract
Computational prediction of microRNAs (miRNAs) is one of the most important approaches in microRNA studies. While validation of the predicted microRNAs' precise sequences is essential for further studies on their biogenesis, evolution, and functions, computational miRNA prediction methods, however, often fail to predict the accurate sequence of the mature miRNA within the precursor at the nucleotide precision level. Here, we depict a highly efficient method for determining the precise sequences of computationally predicted miRNAs. The method combines the generation of miRNA-enriched libraries, with 5'- and 3'-end adaptors being linked to the miRNA molecules, the reverse transcription of small RNAs with an oligo-d(T) anchor primer, two specific 5'- and 3'-miRNA-RACE (miR-RACE) PCR reactions and sequence-directed cloning. The efficiency of this method was demonstrated by the precise sequence validation of computationally predicted miRNAs in citrus, apple, and other fruit crops. Our ongoing research indicates that miR-RACE is also very useful to verify the sequences of putative miRNAs obtained by deep sequencing of small RNA libraries. The protocol of miR-RACE is rapid and can be completed within 2-3 days. miR-RACE should make the bioinformatic prediction of miRNAs more powerful and accurate.

PMID: 25791595 [PubMed - in process]

Extensive microRNA-mediated crosstalk between lncRNAs and mRNAs in mouse embryonic stem cells.

Genome Res. 2015 Mar 19;

Authors: Tan JY, Sirey T, Honti F, Graham B, Piovesan A, Merkenschlager M, Webber C, Ponting CP, Marques AC

Abstract
Recently, a handful of intergenic long noncoding RNAs (lncRNAs) have been shown to compete with mRNAs for binding to miRNAs and to contribute to development and disease. Beyond these reports, little is yet known of the extent and functional consequences of miRNA-mediated regulation of mRNA levels by lncRNAs. To gain further insights into lncRNA-mRNA miRNA-mediated crosstalk, we re-analyzed transcriptome-wide changes induced by the targeted knockdown of over 100 lncRNA transcripts in mouse embryonic stem cells (mESCs). We predicted that on average almost one fifth of the transcript level changes induced by lncRNAs are dependent on miRNAs that are highly abundant in mESCs. We validated these findings experimentally by temporally profiling transcriptome-wide changes in gene expression following the loss of miRNA biogenesis in mESCs. Following the depletion of miRNAs, we found that over 50% of lncRNAs and their miRNA-dependent mRNA targets were up-regulated coordinately, consistent with their interaction being miRNA-mediated. These lncRNAs are preferentially located in the cytoplasm and the response elements for miRNAs they share with their targets have been preserved in mammals by purifying selection. Lastly miRNA-dependent mRNA targets of each lncRNA tended to share common biological functions. Post-transcriptional miRNA-mediated crosstalk between lncRNAs and mRNA, in mESCs, is thus surprisingly prevalent, conserved in mammals and likely to contribute to critical developmental processes.

PMID: 25792609 [PubMed - as supplied by publisher]

Nature Reviews Molecular Cell Biology 16, 205 (2015). doi:10.1038/nrm3976

Author: Kim Baumann

The mechanisms involved in the processing of long non-coding RNAs (lncRNAs) are largely uncharacterized. Dhir et al. now find that transcription termination of lncRNA transcripts containing primary miRNAs (lnc-pri-miRNAs) — which encode 17.5% of human miRNAs — involves cleavage by the Microprocessor complex rather

Nature Reviews Molecular Cell Biology 16, 204 (2015). doi:10.1038/nrm3974

Author: Eytan Zlotorynski

N6-methyladenosine (m6A) is an RNA modification that regulates transcript metabolism. Liu et al. now report that m6A induces structural remodelling in RNAs and that this affects gene expression. They found that a m6A in a hairpin

The evolutionarily conserved gene lin-28 encodes an RNA-binding protein and is an important regulator of the proper temporal succession of several developmental events in both invertebrates and vertebrates. At the cellular level, LIN-28 promotes stemness and proliferation, and inhibits differentiation, a feature best illustrated by its ability to induce pluripotency when ectopically expressed in human fibroblasts in combination with NANOG, OCT4, and SOX2. Mammalian LIN28 functions in part by regulating processing of the let-7 microRNA through a GGAG binding site in the pre-let-7’s distal loop region. However, many human and animal let-7 precursors lack the GGAG binding motif. In order to dissect the molecular mechanisms underlying its biological functions in a living animal, we identified a map of LIN-28 interactions with the transcriptome by in vivo HITS-CLIP in Caenorhabditis elegans. LIN-28 binds a large pool of messenger RNAs, and a substantial fraction of the bona fide LIN-28 targets are involved in aspects of animal development. Furthermore, our data show that LIN-28 regulates the expression of the let-7 microRNA by binding its primary transcript in a previously unknown region, revealing a novel regulatory mechanism.

Giovanni Nigita, Dario Veneziano and Alfredo Ferro

Support staff: Build reward system for ace technicians

Nature 519, 7544 (2015). doi:10.1038/519414e

Author: Eleftherios P. Diamandis

Technical staff are crucial to the smooth running of a research laboratory (see Nature517, 528;10.1038/517528a2015). As largely unsung heroes, they warrant rewards beyond praise and salary increases.A senior technician's duties cover, among other functions, safety, finance, ordering and

Nature advance online publication 25 March 2015. doi:10.1038/nature14346

Authors: Dominique Lauressergues, Jean-Malo Couzigou, Hélène San Clemente, Yves Martinez, Christophe Dunand, Guillaume Bécard & Jean-Philippe Combier

MicroRNAs (miRNAs) are small regulatory RNA molecules that inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs or otherwise inhibiting their translation into proteins. miRNAs are transcribed as much larger primary transcripts (pri-miRNAs), the function of which is not fully understood. Here we show that plant pri-miRNAs contain short open reading frame sequences that encode regulatory peptides. The pri-miR171b of Medicago truncatula and the pri-miR165a of Arabidopsis thaliana produce peptides, which we term miPEP171b and miPEP165a, respectively, that enhance the accumulation of their corresponding mature miRNAs, resulting in downregulation of target genes involved in root development. The mechanism of miRNA-encoded peptide (miPEP) action involves increasing transcription of the pri-miRNA. Five other pri-miRNAs of A. thaliana and M. truncatula encode active miPEPs, suggesting that miPEPs are widespread throughout the plant kingdom. Synthetic miPEP171b and miPEP165a peptides applied to plants specifically trigger the accumulation of miR171b and miR165a, leading to reduction of lateral root development and stimulation of main root growth, respectively, suggesting that miPEPs might have agronomical applications.

RNA-binding proteins in neurodegeneration: Seq and you shall receive.

Trends Neurosci. 2015 Mar 9;

Authors: Nussbacher JK, Batra R, Lagier-Tourenne C, Yeo GW

Abstract
As critical players in gene regulation, RNA binding proteins (RBPs) are taking center stage in our understanding of cellular function and disease. In our era of bench-top sequencers and unprecedented computational power, biological questions can be addressed in a systematic, genome-wide manner. Development of high-throughput sequencing (Seq) methodologies provides unparalleled potential to discover new mechanisms of disease-associated perturbations of RNA homeostasis. Complementary to candidate single-gene studies, these innovative technologies may elicit the discovery of unexpected mechanisms, and enable us to determine the widespread influence of the multifunctional RBPs on their targets. Given that the disruption of RNA processing is increasingly implicated in neurological diseases, these approaches will continue to provide insights into the roles of RBPs in disease pathogenesis.

PMID: 25765321 [PubMed - as supplied by publisher]

The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion.

Annu Rev Biochem. 2015 Mar 11;

Authors: Singh G, Pratt G, Yeo GW, Moore MJ

Abstract
Throughout their lifetimes, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Since the discovery of the first mRNP component more than 40 years ago, what is known as the mRNA interactome now comprises >1,000 proteins. These proteins bind mRNAs in myriad ways with varying affinities and stoichiometries, with many assembling onto nascent RNAs in a highly ordered process during transcription and precursor mRNA (pre-mRNA) processing. The nonrandom distribution of major mRNP proteins observed in transcriptome-wide studies leads us to propose that mRNPs are organized into three major domains loosely corresponding to 5' untranslated regions (UTRs), open reading frames, and 3' UTRs. Moving from the nucleus to the cytoplasm, mRNPs undergo extensive remodeling as they are first acted upon by the nuclear pore complex and then by the ribosome. When not being actively translated, cytoplasmic mRNPs can assemble into large multi-mRNP assemblies or be permanently disassembled and degraded. In this review, we aim to give the reader a thorough understanding of past and current eukaryotic mRNP research. Expected final online publication date for the Annual Review of Biochemistry Volume 84 is June 02, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

PMID: 25784054 [PubMed - as supplied by publisher]

The Expansion of Animal MicroRNA Families Revisited.

Life (Basel). 2015;5(1):905-920

Authors: Hertel J, Stadler PF

Abstract
MicroRNAs are important regulatory small RNAs in many eukaryotes. Due to their small size and simple structure, they are readily innovated de novo. Throughout the evolution of animals, the emergence of novel microRNA families traces key morphological innovations. Here, we use a computational approach based on homology search and parsimony-based presence/absence analysis to draw a comprehensive picture of microRNA evolution in 159 animal species. We confirm previous observations regarding bursts of innovations accompanying the three rounds of genome duplications in vertebrate evolution and in the early evolution of placental mammals. With a much better resolution for the invertebrate lineage compared to large-scale studies, we observe additional bursts of innovation, e.g., in Rhabditoidea. More importantly, we see clear evidence that loss of microRNA families is not an uncommon phenomenon. The Enoplea may serve as a second dramatic example beyond the tunicates. The large-scale analysis presented here also highlights several generic technical issues in the analysis of very large gene families that will require further research.

PMID: 25780960 [PubMed - as supplied by publisher]

Author: Andrew Gaudet

Base-pairing interactions between nucleic acids mediate target recognition in many biological processes. We developed a super-resolution imaging and modeling platform that enabled the in vivo determination of base pairing–mediated target recognition kinetics. We examined a stress-induced bacterial small RNA, SgrS, which induces the degradation of target messenger RNAs (mRNAs). SgrS binds to a primary target mRNA in a reversible and dynamic fashion, and formation of SgrS-mRNA complexes is rate-limiting, dictating the overall regulation efficiency in vivo. Examination of a secondary target indicated that differences in the target search kinetics contribute to setting the regulation priority among different target mRNAs. This super-resolution imaging and analysis approach provides a conceptual framework that can be generalized to other small RNA systems and other target search processes. Authors: Jingyi Fei, Digvijay Singh, Qiucen Zhang, Seongjin Park, Divya Balasubramanian, Ido Golding, Carin K. Vanderpool, Taekjip Ha

Analysis of single molecules in living cells has provided quantitative insights into the kinetics of fundamental biological processes; however, the dynamics of messenger RNA (mRNA) translation have yet to be addressed. We have developed a fluorescence microscopy technique that reports on the first translation events of individual mRNA molecules. This allowed us to examine the spatiotemporal regulation of translation during normal growth and stress and during Drosophila oocyte development. We have shown that mRNAs are not translated in the nucleus but translate within minutes after export, that sequestration within P-bodies regulates translation, and that oskar mRNA is not translated until it reaches the posterior pole of the oocyte. This methodology provides a framework for studying initiation of protein synthesis on single mRNAs in living cells. Authors: James M. Halstead, Timothée Lionnet, Johannes H. Wilbertz, Frank Wippich, Anne Ephrussi, Robert H. Singer, Jeffrey A. Chao

Nature advance online publication 18 March 2015. doi:10.1038/nature14281

Authors: Claudio R. Alarcón, Hyeseung Lee, Hani Goodarzi, Nils Halberg & Sohail F. Tavazoie

The first step in the biogenesis of microRNAs is the processing of primary microRNAs (pri-miRNAs) by the microprocessor complex, composed of the RNA-binding protein DGCR8 and the type III RNase DROSHA. This initial event requires recognition of the junction between the stem and the flanking single-stranded RNA of the pri-miRNA hairpin by DGCR8 followed by recruitment of DROSHA, which cleaves the RNA duplex to yield the pre-miRNA product. While the mechanisms underlying pri-miRNA processing have been determined, the mechanism by which DGCR8 recognizes and binds pri-miRNAs, as opposed to other secondary structures present in transcripts, is not understood. Here we find in mammalian cells that methyltransferase-like 3 (METTL3) methylates pri-miRNAs, marking them for recognition and processing by DGCR8. Consistent with this, METTL3 depletion reduced the binding of DGCR8 to pri-miRNAs and resulted in the global reduction of mature miRNAs and concomitant accumulation of unprocessed pri-miRNAs. In vitro processing reactions confirmed the sufficiency of the N6-methyladenosine (m6A) mark in promoting pri-miRNA processing. Finally, gain-of-function experiments revealed that METTL3 is sufficient to enhance miRNA maturation in a global and non-cell-type-specific manner. Our findings reveal that the m6A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis.

Nature advance online publication 18 March 2015. doi:10.1038/nature14263

Authors: Robert C. Spitale, Ryan A. Flynn, Qiangfeng Cliff Zhang, Pete Crisalli, Byron Lee, Jong-Wha Jung, Hannes Y. Kuchelmeister, Pedro J. Batista, Eduardo A. Torre, Eric T. Kool & Howard Y. Chang

Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2′-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA–protein interactions and N6-methyladenosine (m6A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

Nature advance online publication 18 March 2015. doi:10.1038/nature14280

Authors: Yoichiro Sugimoto, Alessandra Vigilante, Elodie Darbo, Alexandra Zirra, Cristina Militti, Andrea D’Ambrogio, Nicholas M. Luscombe & Jernej Ule

The structure of messenger RNA is important for post-transcriptional regulation, mainly because it affects binding of trans-acting factors. However, little is known about the in vivo structure of full-length mRNAs. Here we present hiCLIP, a biochemical technique for transcriptome-wide identification of RNA secondary structures interacting with RNA-binding proteins (RBPs). Using this technique to investigate RNA structures bound by Staufen 1 (STAU1) in human cells, we uncover a dominance of intra-molecular RNA duplexes, a depletion of duplexes from coding regions of highly translated mRNAs, an unexpected prevalence of long-range duplexes in 3′ untranslated regions (UTRs), and a decreased incidence of single nucleotide polymorphisms in duplex-forming regions. We also discover a duplex spanning 858 nucleotides in the 3′ UTR of the X-box binding protein 1 (XBP1) mRNA that regulates its cytoplasmic splicing and stability. Our study reveals the fundamental role of mRNA secondary structures in gene expression and introduces hiCLIP as a widely applicable method for discovering new, especially long-range, RNA duplexes.

Visualizing transient Watson–Crick-like mispairs in DNA and RNA duplexes

Nature 519, 7543 (2015). doi:10.1038/nature14227

Authors: Isaac J. Kimsey, Katja Petzold, Bharathwaj Sathyamoorthy, Zachary W. Stein & Hashim M. Al-Hashimi

Rare tautomeric and anionic nucleobases are believed to have fundamental biological roles, but their prevalence and functional importance has remained elusive because they exist transiently, in low abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show