Building Robust Transcriptomes with Master Splicing Factors

Coherent splicing networks arise from many discrete splicing decisions regulated in unison. Here, we examine the properties of robust, context-specific splicing networks. We propose that a subset of key splicing regulators, or “master splicing factors,” respond to environmental cues to establish and maintain tissue transcriptomes during development.United States. Public Health Service (RO1-GM34277)United States. Public Health Service (R01-CA133404)United States. Public Health Service (U54-CA112967)National Cancer Institute (U.S.) (P30-CA14051

RNA Bind-n-Seq: Quantitative Assessment of the Sequence and Structural Binding Specificity of RNA Binding Proteins

Specific protein-RNA interactions guide posttranscriptional gene regulation. Here, we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1, and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity.National Science Foundation (U.S.) (0821391

Transcription factor trapping by RNA in gene regulatory elements

Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs.National Institutes of Health (U.S.) (HG002668)Biogen, Inc

Multiple regulatory layers in the establishment of Rbfox2 splicing networks

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2014.Cataloged from PDF version of thesis. Vita.Includes bibliographical references.Regulated alternative splicing is mediated by RNA binding proteins (RBPs) recognizing short sequence motifs in nascent transcripts. The Rbfox RBPs are highly conserved splicing factors that regulate tissue-specific exon inclusion by binding the RNA motif UGCAUG. We sought to comprehensively define the Rbfox2 splicing regulatory network and discover determinants of Rbfox2 regulation in mouse embryonic stem cells. We uncovered fundamental principles in the mechanistic aspects of Rbfox-dependent splicing and in the systems-level regulation of interconnected splicing networks. Using high-resolution iCLIP and RNAseq, we identified many Rbfox2-mediated proteincoding splicing events and nearly 300 additional events, in particular those within RBPs, that are coupled to nonsense-mediated mRNA decay (NMD). Regulation of NMD-coupled splicing by Rbfox2 alters gene expression of autoregulated RBPs and hundreds of additional genes. These observations place Rbfox2 upstream of a large network of direct and indirect splicing changes and offer an explanation as to how autoregulated gene expression can be modulated. We describe a validation of RNA Bind-n-Seq, a novel in vitro technique for analyzing RNA-protein interactions. We found a secondary Rbfox2 motif, GCACG, to be functional in splicing regulation in addition to the consensus UGCAUG and observed a preference for Rbfox2 binding to unstructured sequences. These findings provide a foundation for establishing the critical determinants of functional cis elements in splicing regulation. We also investigated mechanisms of co-transcriptional splicing regulation by Rbfox2. Using chromatin immunoprecipitation, we found that Rbfox2 is recruited early in the transcription cycle to active promoters and transcriptional enhancers, likely via interaction and co-transcriptional tracking with RNA polymerase II. Modulation of chromatin structure alters Rbfox2-dependent splicing activity, supporting the emerging model that the chromatin environment influences exon choice. Our analyses of Rbfox2 activity in mouse embryonic stem cells reveal hundreds of previously unknown splicing targets involved in diverse biological functions. In particular, we introduce a novel concept in splicing regulation whereby changes in the expression of one splicing factor induce a cascade of secondary and perhaps tertiary splicing changes through cross-regulation of RBPs. This model positions RBPs at a critical node in the establishment, reinforcement, and alteration of tissue transcriptomes during mammalian development.by Mohini Jangi.Ph. D