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

Abstract

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