Widespread intron retention in mammals functionally tunes transcriptomes

© 2014 Braunschweig et al.; Published by Cold Spring Harbor Laboratory Press. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.Alternative splicing (AS) of precursor RNAs is responsible for greatly expanding the regulatory and functional capacity of eukaryotic genomes. Of the different classes of AS, intron retention (IR) is the least well understood. In plants and unicellular eukaryotes, IR is the most common form of AS, whereas in animals, it is thought to represent the least prevalent form. Using high-coverage poly(A)(+) RNA-seq data, we observe that IR is surprisingly frequent in mammals, affecting transcripts from as many as three-quarters of multiexonic genes. A highly correlated set of cis features comprising an "IR code" reliably discriminates retained from constitutively spliced introns. We show that IR acts widely to reduce the levels of transcripts that are less or not required for the physiology of the cell or tissue type in which they are detected. This "transcriptome tuning" function of IR acts through both nonsense-mediated mRNA decay and nuclear sequestration and turnover of IR transcripts. We further show that IR is linked to a cross-talk mechanism involving localized stalling of RNA polymerase II (Pol II) and reduced availability of spliceosomal components. Collectively, the results implicate a global checkpoint-type mechanism whereby reduced recruitment of splicing components coupled to Pol II pausing underlies widespread IR-mediated suppression of inappropriately expressed transcripts.This work was supported by grants from the Canadian Institutes of Health Research and Canadian Cancer Society (B.J.B.); EMBO long-term fellowships (U.B. and T.G.-P.); Human Frontier Science Program Organization long-term fellowships (U.B. and M.I.); an OSCI fellowship (T.G.-P.); CIHR postdoctoral and Marie Curie IOF fellowships (N.L.B.-M.); and an NSERC studentship (E.N.).info:eu-repo/semantics/publishedVersio

MBNL proteins repress ES-cell-specific alternative splicing and reprogramming

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators. Alternative splicing represents a widely acting mode of gene regulation, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.Canadian Institutes of Health Research (grant)Ontario Research FoundationStem Cell Network (Canada)National Institutes of Health (U.S.) (grant R33MH087908)University of Toronto (Open Fellowship)Ontario Stem Cell InitiativeHuman Frontier Science Program (Strasbourg, France)European Union (Marie Curie Actions

MBNL proteins repress ES-cellspecific alternative splicing and reprogramming. Nature 498: 241–245. doi: 10.1038/nature12270 PMID: 23739326

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators A core set of transcription factors that includes OCT4 (also called POU5F1), NANOG and SOX2, together with specific microRNAs and long non-coding RNAs, control the expression of genes required for the establishment and maintenance of ES-cell pluripotency To identify such factors, we used high-throughput RNA sequencing (RNA-seq) data to define human and mouse cassette alternative exons that are differentially spliced between ES cells and induced pluripotent stem cells (iPSCs), and diverse differentiated cells and tissues, referred to below as 'ES-cell-differential alternative splicing'. A splicing code analysis 17 was then performed to identify cis-elements that may promote or repress these exons. The RNA-seq data used to profile alternative splicing were also used to detect human and mouse splicing factor genes that are differentially expressed between ES cells/iPSCs and non-ES cells/tissues. By integrating these data sources, we sought to identify differentially expressed splicing regulators with defined binding sites that match cis-elements predicted by the code analysis to function in ES-cell-differential alternative splicing. We identified 181 human and 103 mouse ES-cell-differential alternative splicing events, with comparable proportions of exons that are $25% more included or more skipped in ES cells versus the other profiled cells and tissues 216 ; hypergeometric test). The human and mouse ES-cell-differential alternative splicing events are significantly enriched in genes associated with the cytoskeleton (for example, DST, ADD3), plasma membrane (for example, DNM2, ITGA6) and kinase activity (for example, CASK, MARK2 and MAP2K7) (Supplementary The splicing code analysis revealed that motifs corresponding to consensus binding sites of the conserved MBNL proteins are the most strongly associated with ES-cell-differential alternative splicing in human and mouse. The presence of MBNL motifs in downstream flanking intronic sequences is associated with exon skipping in ES cells, whereas their presence in upstream flanking intronic sequences is associated with exon inclusion in ES cells From RNA-seq expression profiling of 221 known or putative splicing factors, 11 genes showed significant differential expression between human ES cells/iPSCs and other cells and tissues (Bonferronicorrected P , 0.05, Wilcoxon rank-sum test