Hyeshik Chang

Related Articles

Sequencing degraded RNA addressed by 3' tag counting.

PLoS One. 2014;9(3):e91851

Authors: Sigurgeirsson B, Emanuelsson O, Lundeberg J

Abstract
RNA sequencing has become widely used in gene expression profiling experiments. Prior to any RNA sequencing experiment the quality of the RNA must be measured to assess whether or not it can be used for further downstream analysis. The RNA integrity number (RIN) is a scale used to measure the quality of RNA that runs from 1 (completely degraded) to 10 (intact). Ideally, samples with high RIN (> 8) are used in RNA sequencing experiments. RNA, however, is a fragile molecule which is susceptible to degradation and obtaining high quality RNA is often hard, or even impossible when extracting RNA from certain clinical tissues. Thus, occasionally, working with low quality RNA is the only option the researcher has. Here we investigate the effects of RIN on RNA sequencing and suggest a computational method to handle data from samples with low quality RNA which also enables reanalysis of published datasets. Using RNA from a human cell line we generated and sequenced samples with varying RINs and illustrate what effect the RIN has on the basic procedure of RNA sequencing; both quality aspects and differential expression. We show that the RIN has systematic effects on gene coverage, false positives in differential expression and the quantification of duplicate reads. We introduce 3' tag counting (3TC) as a computational approach to reliably estimate differential expression for samples with low RIN. We show that using the 3TC method in differential expression analysis significantly reduces false positives when comparing samples with different RIN, while retaining reasonable sensitivity.

PMID: 24632678 [PubMed - indexed for MEDLINE]

Related Articles

Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes.

Genes Dev. 2014 Nov 15;28(22):2498-517

Authors: Lykke-Andersen S, Chen Y, Ardal BR, Lilje B, Waage J, Sandelin A, Jensen TH

Abstract
Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities on endogenous substrates has remained unresolved. Here we used concurrent transcriptome-wide identification of NMD substrates and their 5'-3' decay intermediates to establish that SMG6-catalyzed endonucleolysis widely initiates the degradation of human nonsense RNAs, whereas decapping is used to a lesser extent. We also show that a large proportion of genes hosting snoRNAs in their introns produce considerable amounts of NMD-sensitive splice variants, indicating that these RNAs are merely by-products of a primary snoRNA production process. Additionally, transcripts from genes encoding multiple snoRNAs often yield alternative transcript isoforms that allow for differential expression of individual coencoded snoRNAs. Based on our findings, we hypothesize that snoRNA host genes need to be highly transcribed to accommodate high levels of snoRNA production and that the expression of individual snoRNAs and their cognate spliced RNA can be uncoupled via alternative splicing and NMD.

PMID: 25403180 [PubMed - in process]

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Transcriptome-wide Variability in Single Embryonic Development Cells.

Sci Rep. 2014;4:7137

Authors: Piras V, Tomita M, Selvarajoo K

Abstract
Molecular heterogeneity of individual molecules within single cells has been recently shown to be crucial for cell fate diversifications. However, on a global scale, the effect of molecular variability for embryonic developmental stages is largely underexplored. Here, to understand the origins of transcriptome-wide variability of oocytes to blastocysts in human and mouse, we examined RNA-Seq datasets. Evaluating Pearson correlation, Shannon entropy and noise patterns (η(2) vs. μ), our investigations reveal a phase transition from low to saturating levels of diversity and variability of transcriptome-wide expressions through the development stages. To probe the observed behaviour further, we utilised a stochastic transcriptional model to simulate the global gene expressions pattern for each development stage. From the model, we concur that transcriptome-wide regulation initially begins from 2-cell stage, and becomes strikingly variable from 8-cell stage due to amplification and quantal transcriptional activity.

PMID: 25409746 [PubMed - as supplied by publisher]

A Variety of Dicer Substrates in Human and C. elegans.

Cell. 2014 Nov 20;159(5):1153-67

Authors: Rybak-Wolf A, Jens M, Murakawa Y, Herzog M, Landthaler M, Rajewsky N

Abstract
The endoribonuclease Dicer is known for its central role in the biogenesis of eukaryotic small RNAs/microRNAs. Despite its importance, Dicer target transcripts have not been directly mapped. Here, we apply biochemical methods to human cells and C. elegans and identify thousands of Dicer-binding sites. We find known and hundreds of additional miRNAs with high sensitivity and specificity. We also report structural RNAs, promoter RNAs, and mitochondrial transcripts as Dicer targets. Interestingly, most Dicer-binding sites reside on mRNAs/lncRNAs and are not significantly processed into small RNAs. These passive sites typically harbor small, Dicer-bound hairpins within intact transcripts and generally stabilize target expression. We show that passive sites can sequester Dicer and reduce microRNA expression. mRNAs with passive sites were in human and worm significantly associated with processing-body/granule function. Together, we provide the first transcriptome-wide map of Dicer targets and suggest conserved binding modes and functions outside of the miRNA pathway.

PMID: 25416952 [PubMed - in process]

Publication date: 18 December 2014
Source:Molecular Cell, Volume 56, Issue 6
Author(s): Simon R.V. Knott , Ashley R. Maceli , Nicolas Erard , Kenneth Chang , Krista Marran , Xin Zhou , Assaf Gordon , Osama El Demerdash , Elvin Wagenblast , Sun Kim , Christof Fellmann , Gregory J. Hannon
The strength of conclusions drawn from RNAi-based studies is heavily influenced by the quality of tools used to elicit knockdown. Prior studies have developed algorithms to design siRNAs. However, to date, no established method has emerged to identify effective shRNAs, which have lower intracellular abundance than transfected siRNAs and undergo additional processing steps. We recently developed a multiplexed assay for identifying potent shRNAs and used this method to generate ∼250,000 shRNA efficacy data points. Using these data, we developed shERWOOD, an algorithm capable of predicting, for any shRNA, the likelihood that it will elicit potent target knockdown. Combined with additional shRNA design strategies, shERWOOD allows the ab initio identification of potent shRNAs that specifically target the majority of each gene’s multiple transcripts. We validated the performance of our shRNA designs using several orthogonal strategies and constructed genome-wide collections of shRNAs for humans and mice based on our approach.

Graphical abstract

Teaser

The identification of potent shRNAs is difficult, therefore limiting their utility for identifying gene targets. Knott et al. have developed a computational algorithm capable of predicting potent shRNAs for any target sequence. shRNAs selected with this algorithm were found to be more efficacious than those in currently available libraries.

Publication date: December 2014
Source:Trends in Cell Biology, Volume 24, Issue 12
Author(s): Jane A. Skok
Scientists are by nature a determined, dedicated breed and do not give up easily even when confronted with seemingly insurmountable obstacles. So why would anyone who has embarked along this career path choose to take a break from it, and can one really return to a successful academic career after this break?

Background: Micro-RNAs (miRNA) are attributed to the systems biological role of a regulatory mechanism of the expression of protein coding genes. Research has identified miRNAs dysregulations in several but distinct pathophysiological processes, which hints at distinct systems-biology functions of miRNAs. The present analysis approached the role of miRNAs from a genomics perspective and assessed the biological roles of 2954 genes and 788 human miRNAs, which can be considered to interact, based on empirical evidence and computational predictions of miRNA versus gene interactions. Results: From a genomics perspective, the biological processes in which the genes that are influenced by miRNAs are involved comprise of six major topics comprising biological regulation, cellular metabolism, information processing, development, gene expression and tissue homeostasis. The usage of this knowledge as a guidance for further research is sketched for two genetically defined functional areas: cell death and gene expression. Results suggest that the latter points to a fundamental role of miRNAs consisting of hyper-regulation of gene expression, i.e., the control of the expression of such genes which control specifically the expression of genes. Conclusions: Laboratory research identified contributions of miRNA regulation to several distinct biological processes. The present analysis transferred this knowledge to a systems-biology level. A comprehensible and precise description of the biological processes in which the genes that are influenced by miRNAs are notably involved could be made. This knowledge can be employed to guide future research concerning the biological role of miRNA (dys-) regulations. The analysis also suggests that miRNAs especially control the expression of genes that control the expression of genes.

Background: MicroRNAs are a class of short non-coding RNAs derived from either cellular or viral transcripts that act post-transcriptionally to regulate mRNA stability and translation. In recent days, increasing numbers of miRNAs have been shown to be involved in the development and progression of a variety of diseases. We, therefore, intend to enumerate miRNA targets in several known disease classes to explore the degree of miRNA regulations on them which is unexplored till date. Results: Here, we noticed that miRNA hits in cancer genes are remarkably higher than other diseases in human. Our observation suggests that UTRs and the transcript length of cancer related genes have a significant contribution in higher susceptibility to miRNA regulation. Moreover, gene duplication, mRNA stability, AREScores and evolutionary rate were likely to have implications for more miRNA targeting on cancer genes. Consequently, the regression analysis have confirmed that the AREScores plays most important role in detecting miRNA targets on disease genes. Interestingly, we observed that epigenetic modifications like CpG methylation and histone modification are less effective than miRNA regulations in controlling the gene expression of cancer genes. Conclusions: The intrinsic properties of cancer genes studied here, for higher miRNA targeting will enhance the knowledge on cancer gene regulation.

Nature Genetics 46, 1311 (2014). doi:10.1038/ng.3142

Authors: Leighton J Core, André L Martins, Charles G Danko, Colin T Waters, Adam Siepel & John T Lis

Dissecting noncoding and pathogen RNA-protein interactomes.

RNA. 2014 Nov 19;

Authors: Flynn RA, Martin L, Spitale RC, Do BT, Sagan SM, Zarnegar B, Qu K, Khavari PA, Quake SR, Sarnow P, Chang HY

Abstract
RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

PMID: 25411354 [PubMed - as supplied by publisher]

Breast cancer cells secrete exosomes carrying microRNA-processing proteins that promote malignant transformation in normal cells.

Mouse-to-human genomic comparisons illuminate conserved transcriptional programs despite regulatory rewiring. Authors: Jeff Vierstra, Eric Rynes, Richard Sandstrom, Miaohua Zhang, Theresa Canfield, R. Scott Hansen, Sandra Stehling-Sun, Peter J. Sabo, Rachel Byron, Richard Humbert, Robert E. Thurman, Audra K. Johnson, Shinny Vong, Kristen Lee, Daniel Bates, Fidencio Neri, Morgan Diegel, Erika Giste, Eric Haugen, Douglas Dunn, Matthew S. Wilken, Steven Josefowicz, Robert Samstein, Kai-Hsin Chang, Evan E. Eichler, Marella De Bruijn, Thomas A. Reh, Arthur Skoultchi, Alexander Rudensky, Stuart H. Orkin, Thalia Papayannopoulou, Piper M. Treuting, Licia Selleri, Rajinder Kaul, Mark Groudine, M. A. Bender, John A. Stamatoyannopoulos

Article

A large portion of the transcribed genome—such as introns and noncoding RNAs—is believed to not be translated into protein products. Here, the authors provide evidence for the existence of regulated peptide products that are translated from transcribed sequences generally characterized as noncoding.

Nature Communications doi: 10.1038/ncomms6429

Authors: Sudhakaran Prabakaran, Martin Hemberg, Ruchi Chauhan, Dominic Winter, Ry Y. Tweedie-Cullen, Christian Dittrich, Elizabeth Hong, Jeremy Gunawardena, Hanno Steen, Gabriel Kreiman, Judith A. Steen

Article

Small RNA-binding ARGONAUTE proteins are important regulators of gene silencing and plant development. Here Zhang et al. identify conserved motifs in Arabidopsis ARGONAUTE proteins that determine binding to specific microRNA duplex structures.

Nature Communications doi: 10.1038/ncomms6468

Authors: Xiaoming Zhang, DongDong Niu, Alberto Carbonell, Airong Wang, Angel Lee, Vinnary Tun, Zonghua Wang, James C. Carrington, Chia-en A. Chang, Hailing Jin

Article

The use of alternative polyadenylation sites can potentially result in mRNA being more or less susceptible to interaction with modulators of translation or stability. Here Gruber et al . find that the shortening of 3′UTRs observed in proliferating T cells does not significantly impact protein abundance.

Nature Communications doi: 10.1038/ncomms6465

Authors: Andreas R. Gruber, Georges Martin, Philipp Müller, Alexander Schmidt, Andreas J. Gruber, Rafal Gumienny, Nitish Mittal, Rajesh Jayachandran, Jean Pieters, Walter Keller, Erik van Nimwegen, Mihaela Zavolan

Article

The predictable assembly of DNA makes it a useful scaffold for creating pathways to guide nanotransport systems. Here the authors use reversible covalent capture of DNA by quinone methide generation, as well as diffusion along the nucleophilic surface of DNA to guide migration.

Nature Communications doi: 10.1038/ncomms6591

Authors: Fazel Fakhari, Steven E. Rokita

Publication date: January 2015
Source:Trends in Biochemical Sciences, Volume 40, Issue 1
Author(s): Samuel H. Payne
Transcriptomic, proteomic, and metabolomic measurements are revolutionizing the way we model and predict cellular behavior, and multi-omic comparisons are being published with increased regularity. Some have expected a trivial and predictable correlation between mRNA and protein; however, the manifest complexity of biological regulation suggests a more nuanced relationship. Indeed, observing this lack of strict correlation provides clues for new research topics, and has the potential for transformative biological insight.

Background: Prostate cancer (PrCa) is the most commonly diagnosed cancer in men in the world. Despite the fact that a large number of its genes have been investigated, its etiology remains poorly understood. Furthermore, most PrCa candidate genes have not been rigorously replicated, and the methods by which they biologically function in PrCa remain largely unknown. Results: Aiming to identify key players in the complex prostate cancer system, we reconstructed PrCa co-expressed modules within functional gene sets defined by the Gene Ontology (GO) annotation (biological process, GO_BP). We primarily identified 118 GO_BP terms that were well-preserved between two independent gene expression datasets and a consequent 55 conserved co-expression modules within them. Five modules were then found to be significantly enriched with PrCa candidate genes collected from expression Quantitative Trait Loci (eQTL), somatic copy number alteration (SCNA), somatic mutation data, or prognostic analyses. Specifically, two transcription factors (TFs) (NFAT and SP1) and three microRNAs (hsa-miR-19a, hsa-miR-15a, and hsa-miR-200b) regulating these five candidate modules were found to be critical to the development of PrCa. Conclusions: Collectively, our results indicated that genes with similar functions may play important roles in disease through co-expression, and modules with different functions could be regulated by similar genetic components, such as TFs and microRNAs, in a synergistic manner.

Nature advance online publication 19 November 2014. doi:10.1038/nature14010

Authors: Shifeng Xue, Siqi Tian, Kotaro Fujii, Wipapat Kladwang, Rhiju Das & Maria Barna

Conservation of trans-acting circuitry during mammalian regulatory evolution

Nature 515, 7527 (2014). doi:10.1038/nature13972

Authors: Andrew B. Stergachis, Shane Neph, Richard Sandstrom, Eric Haugen, Alex P. Reynolds, Miaohua Zhang, Rachel Byron, Theresa Canfield, Sandra Stelhing-Sun, Kristen Lee, Robert E. Thurman, Shinny Vong, Daniel Bates, Fidencio Neri, Morgan Diegel, Erika Giste, Douglas Dunn, Jeff Vierstra, R. Scott Hansen, Audra K. Johnson, Peter J. Sabo, Matthew S. Wilken, Thomas A. Reh, Piper M. Treuting, Rajinder Kaul, Mark Groudine, M. A. Bender, Elhanan Borenstein & John A. Stamatoyannopoulos

The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic

A comparative encyclopedia of DNA elements in the mouse genome

Nature 515, 7527 (2014). doi:10.1038/nature13992

Authors: Feng Yue, Yong Cheng, Alessandra Breschi, Jeff Vierstra, Weisheng Wu, Tyrone Ryba, Richard Sandstrom, Zhihai Ma, Carrie Davis, Benjamin D. Pope, Yin Shen, Dmitri D. Pervouchine, Sarah Djebali, Robert E. Thurman, Rajinder Kaul, Eric Rynes, Anthony Kirilusha, Georgi K. Marinov, Brian A. Williams, Diane Trout, Henry Amrhein, Katherine Fisher-Aylor, Igor Antoshechkin, Gilberto DeSalvo, Lei-Hoon See, Meagan Fastuca, Jorg Drenkow, Chris Zaleski, Alex Dobin, Pablo Prieto, Julien Lagarde, Giovanni Bussotti, Andrea Tanzer, Olgert Denas, Kanwei Li, M. A. Bender, Miaohua Zhang, Rachel Byron, Mark T. Groudine, David McCleary, Long Pham, Zhen Ye, Samantha Kuan, Lee Edsall, Yi-Chieh Wu, Matthew D. Rasmussen, Mukul S. Bansal, Manolis Kellis, Cheryl A. Keller, Christapher S. Morrissey, Tejaswini Mishra, Deepti Jain, Nergiz Dogan, Robert S. Harris, Philip Cayting, Trupti Kawli, Alan P. Boyle, Ghia Euskirchen, Anshul Kundaje, Shin Lin, Yiing Lin, Camden Jansen, Venkat S. Malladi, Melissa S. Cline, Drew T. Erickson, Vanessa M. Kirkup, Katrina Learned, Cricket A. Sloan, Kate R. Rosenbloom, Beatriz Lacerda de Sousa, Kathryn Beal, Miguel Pignatelli, Paul Flicek, Jin Lian, Tamer Kahveci, Dongwon Lee, W. James Kent, Miguel Ramalho Santos, Javier Herrero, Cedric Notredame, Audra Johnson, Shinny Vong, Kristen Lee, Daniel Bates, Fidencio Neri, Morgan Diegel, Theresa Canfield, Peter J. Sabo, Matthew S. Wilken, Thomas A. Reh, Erika Giste, Anthony Shafer, Tanya Kutyavin, Eric Haugen, Douglas Dunn, Alex P. Reynolds, Shane Neph, Richard Humbert, R. Scott Hansen, Marella De Bruijn, Licia Selleri, Alexander Rudensky, Steven Josefowicz, Robert Samstein, Evan E. Eichler, Stuart H. Orkin, Dana Levasseur, Thalia Papayannopoulou, Kai-Hsin Chang, Arthur Skoultchi, Srikanta Gosh, Christine Disteche, Piper Treuting, Yanli Wang, Mitchell J. Weiss, Gerd A. Blobel, Xiaoyi Cao, Sheng Zhong, Ting Wang, Peter J. Good, Rebecca F. Lowdon, Leslie B. Adams, Xiao-Qiao Zhou, Michael J. Pazin, Elise A. Feingold, Barbara Wold, James Taylor, Ali Mortazavi, Sherman M. Weissman, John A. Stamatoyannopoulos, Michael P. Snyder, Roderic Guigo, Thomas R. Gingeras, David M. Gilbert, Ross C. Hardison, Michael A. Beer & Bing Ren

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the

Principles of regulatory information conservation between mouse and human

Nature 515, 7527 (2014). doi:10.1038/nature13985

Authors: Yong Cheng, Zhihai Ma, Bong-Hyun Kim, Weisheng Wu, Philip Cayting, Alan P. Boyle, Vasavi Sundaram, Xiaoyun Xing, Nergiz Dogan, Jingjing Li, Ghia Euskirchen, Shin Lin, Yiing Lin, Axel Visel, Trupti Kawli, Xinqiong Yang, Dorrelyn Patacsil, Cheryl A. Keller, Belinda Giardine, The Mouse ENCODE Consortium, Anshul Kundaje, Ting Wang, Len A. Pennacchio, Zhiping Weng, Ross C. Hardison & Michael P. Snyder

To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human–mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here

Nature Chemical Biology 10, 1049 (2014). doi:10.1038/nchembio.1655

Authors: Richard I McDonald, John P Guilinger, Shankar Mukherji, Edward A Curtis, Won I Lee & David R Liu

Publication date: December 2014
Source:Trends in Genetics, Volume 30, Issue 12
Author(s): Adriana Estela Sedeño-Cortés , Paul Pavlidis
Gene-set analysis (GSA) (‘enrichment’) is a popular approach for the interpretation of genome-wide association studies (GWASs). GSA is most commonly applied to the analysis of transcriptomes, but from the outset it has been considered useful for any study that provides rankings or ‘hit lists’ of genes. The recent review by Mooney et al. [1] is a valuable resource for geneticists wishing to apply GSA to the output of GWASs. Here we describe some additional points of practical importance if the methods are to be applied and interpreted soundly.

Publication date: 4 December 2014
Source:Molecular Cell, Volume 56, Issue 5
Author(s): Mihye Lee , Yeon Choi , Kijun Kim , Hua Jin , Jaechul Lim , Tuan Anh Nguyen , Jihye Yang , Minsun Jeong , Antonio J. Giraldez , Hui Yang , Dinshaw J. Patel , V. Narry Kim
Early development depends heavily on accurate control of maternally inherited mRNAs, and yet it remains unknown how maternal microRNAs are regulated during maternal-to-zygotic transition (MZT). We here find that maternal microRNAs are highly adenylated at their 3′ ends in mature oocytes and early embryos. Maternal microRNA adenylation is widely conserved in fly, sea urchin, and mouse. We identify Wispy, a noncanonical poly(A) polymerase, as the enzyme responsible for microRNA adenylation in flies. Knockout of wispy abrogates adenylation and results in microRNA accumulation in eggs, whereas overexpression of Wispy increases adenylation and reduces microRNA levels in S2 cells. Wispy interacts with Ago1 through protein-protein interaction, which may allow the effective and selective adenylation of microRNAs. Thus, adenylation may contribute to the clearance of maternally deposited microRNAs during MZT. Our work provides mechanistic insights into the regulation of maternal microRNAs and illustrates the importance of RNA tailing in development.

Graphical abstract

Teaser

Lee et al. find that maternally inherited miRNAs are adenylated in oocytes and early embryos of several organisms. Wispy, a noncanonical poly(A) polymerase, mediates miRNA adenylation and reduces miRNA abundance, contributing to clearance of maternal miRNAs from early embryos.

Publication date: 6 November 2014
Source:Molecular Cell, Volume 56, Issue 3
Author(s): Andrew D. Bosson , Jesse R. Zamudio , Phillip A. Sharp
Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ∼3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition in vivo.

Graphical abstract

Teaser

Bosson et al. estimate the total copies per cell of all miRNA and corresponding targets to address the debated competing endogenous RNA (ceRNA) hypothesis. They show that only miRNAs with low miRNA:target ratios are susceptible to regulation by large induction of high-affinity ceRNAs.

Publication date: 4 December 2014
Source:Molecular Cell, Volume 56, Issue 5
Author(s): Wei Wang , Mayu Yoshikawa , Bo W. Han , Natsuko Izumi , Yukihide Tomari , Zhiping Weng , Phillip D. Zamore
PIWI-interacting RNAs (piRNAs) silence transposons in animal germ cells. PIWI proteins bind and amplify piRNAs via the “Ping-Pong” pathway. Because PIWI proteins cleave RNAs between target nucleotides t10 and t11—the nucleotides paired to piRNA guide positions g10 and g11—the first ten nucleotides of piRNAs participating in the Ping-Pong amplification cycle are complementary. Drosophila piRNAs bound to the PIWI protein Aubergine typically begin with uridine (1U), while piRNAs bound to Argonaute3, which are produced by Ping-Pong amplification, often have adenine at position 10 (10A). The Ping-Pong model proposes that the 10A is a consequence of 1U. We find that 10A is not caused by 1U. Instead, fly Aubergine as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs; during Ping-Pong amplification, this t1A subsequently becomes the g10A of a piRNA bound to Argonaute3.

Graphical abstract

Teaser

Aubergine-bound piRNAs typically begin with uracil (g1U) and bind RNAs bearing an adenine (t1A) across from that nucleotide; g1U was presumed to pair with t1A. Instead, Wang et al. find that Aubergine itself—not base pairing—selects for the target t1A, which subsequently becomes the hallmark g10A of Ago3-bound piRNAs.

Background: Ribosome profiling (ribo-seq) provides experimental data on the density of elongating or initiating ribosomes at the whole transcriptome level that can be potentially used for estimating absolute levels of translation initiation at individual Translation Initiation Sites (TISs). These absolute levels depend on the mutual organisation of TISs within individual mRNAs. For example, according to the leaky scanning model of translation initiation in eukaryotes, a strong TIS downstream of another strong TIS is unlikely to be productive, since only a few scanning ribosomes would be able to reach the downstream TIS. In order to understand the dependence of translation initiation efficiency on the surrounding nucleotide context, it is important to estimate the strength of TISs independently of their mutual organisation, i.e. to estimate with what probability a ribosome would initiate at a particular TIS. Results: We designed a simple computational approach for estimating the probabilities of ribosomes initiating at individual start codons using ribosome profiling data. The method is based on the widely accepted leaky scanning model of translation initiation in eukaryotes which postulates that scanning ribosomes may skip a start codon if the initiation context is unfavourable and continue on scanning. We tested our approach on three independent ribo-seq datasets obtained in mammalian cultured cells. Conclusions: Our results suggested that the method successfully discriminates between weak and strong TISs and that the majority of numerous non-AUG TISs reported recently are very weak. Therefore the high frequency of non-AUG TISs observed in ribosome profiling experiments is due to their proximity to mRNA 5′-ends rather than their strength. Detectable translation initiation at non-AUG codons downstream of AUG codons is comparatively infrequent. The leaky scanning method will be useful for the characterization of differences in start codon selection between tissues, developmental stages and in response to stress conditions.