Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors

Abstract

Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.

Regulation of alternative mRNA splicing: old players and new perspectives

“…Splice site selection was affected not only by proteins involved in early splice site recognition (U1 and U2 snRNP) but also proteins involved in later stages of spliceosome formation and splicing catalysis . These results were subsequently corroborated by a mouse‐based study that assayed a comparable range of alternative splicing events, but included knockdown of a wider range of proteins . Previous single‐gene studies have implicated the expression level of core spliceosomal proteins in alternative splicing.…”

“…Recent high-throughput screens have revealed that individual splicing events are jointly controlled by an extensive range of proteins spanning multiple regulatory layers within the cell [14,15]. Splicing of a given mRNA is also affected by the rate of RNA polymerase II (RNAPII) elongation along the gene body [16] and the epigenetic environment of the gene locus [17].…”

“…Enhancer and silencer motifs may stimulate or inhibit splicing, respectively, at specific splice sites , mediated by RNA‐binding proteins (RBPs) acting as splicing enhancers or repressors. Recent high‐throughput screens have revealed that individual splicing events are jointly controlled by an extensive range of proteins spanning multiple regulatory layers within the cell . Splicing of a given mRNA is also affected by the rate of RNA polymerase II (RNAPII) elongation along the gene body and the epigenetic environment of the gene locus .…”

“…Exploring the regulation of alternative splicing and the underlying mechanisms is therefore critical to our understanding of the fundamental biology of both healthy and malignant cells.In the classic view of splicing regulation, the main determinants of alternative splicing are cis-acting sequence features within the pre-mRNA [9], and a range of regulatory splicing factor proteins acting in trans [10,11]. Enhancer and silencer motifs may stimulate or inhibit splicing, respectively, at specific splice sites [12,13], mediated by RNA-binding proteins (RBPs) acting as splicing enhancers or repressors.Recent high-throughput screens have revealed that individual splicing events are jointly controlled by an extensive range of proteins spanning multiple regulatory layers within the cell [14,15]. Splicing of a given mRNA is also affected by the rate of RNA polymerase II (RNAPII) elongation along the gene body [16] and the epigenetic environment of the gene locus [17].…”

Pre-mRNA Splicing Modulation by Antisense Oligonucleotides

“…Also, transcription of protein-coding genes is controlled by local chromatin structure which in turn is affected by multiple factors, including nucleosome positioning, histone modifications, and DNA methylation. Therefore, factors involved in the abovementioned processes have potential to effect splicing outcomes (8–11). Use of minigenes has been very helpful in deciphering the combinatorial control executed by various cis -elements (12).…”