Published online 20 February 2016 Nucleic Acids Research, 2016, Vol. 44, No. 5 2283–2297 doi: 10.1093/nar/gkw088 ␣CP binding to a cytosine-rich subset of polypyrimidine tracts drives a novel pathway of cassette exon splicing in the mammalian transcriptome Xinjun Ji1,*,†, Juw Won Park2,3,4,†, Emad Bahrami-Samani2, Lan Lin2, Christopher Duncan-Lewis1, Gordon Pherribo1,YiXing2,* and Stephen A. Liebhaber1,5,*
1Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA, 2Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA, 3Department of Computer Engineering and Computer Science, University of Louisville, Louisville, KY 40292, USA, 4KBRIN Bioinformatics Core, University of Louisville, Louisville, KY 40202, USA and 5Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
Received September 01, 2015; Revised January 27, 2016; Accepted February 03, 2016
ABSTRACT INTRODUCTION Alternative splicing (AS) is a robust generator of RNA splicing is a highly dynamic process that involves as mammalian transcriptome complexity. Splice site many as 200 protein factors interacting with target sites on a specification is controlled by interactions of cis- PolII transcript. While many of these RNA-protein (RNP) acting determinants on a transcript with specific interactions have been described in detail, the broad array RNA binding proteins. These interactions are fre- of alternative splicing (AS) events detected in mammalian cells (1,2) and the large number of RNA binding proteins quently localized to the intronic U-rich polypyrimi- (RBPs) encoded by the mammalian genome, suggest that dine tracts (PPT) located 5 to the majority of splice numerous additional determinants of splicing controls re- ␣ acceptor junctions. CPs (also referred to as polyC- main to be identified and characterized (3–7). Many of the binding proteins (PCBPs) and hnRNPEs) comprise a relevant RBPs (at least 141) compose core components of subset of KH-domain proteins with high affinity and the mammalian spliceosome complexes (3,8–10) that inter- specificity for C-rich polypyrimidine motifs. Here, we act with splice donor and splice acceptor regions and cat- demonstrate that ␣CPs promote the splicing of a de- alyze intron excision and exon ligation (8). The ‘strength’ of fined subset of cassette exons via binding to a C- a splice acceptor site is impacted by the assembly of RNP complexes at a polypyrimidine tract (PPT) located imme- rich subset of polypyrimidine tracts located 5 to the ␣CP-enhanced exonic segments. This enhancement diately 5 of the AG splicing acceptor site. This PPT char- of splice acceptor activity is linked to interactions of acteristically consists of a loosely defined U-rich sequence with interspersed C residues (8,11–14). The splicing factor ␣CPs with the U2 snRNP complex and may be medi- U2AF65 binds directly to this U-rich PPT and recruits its ated by cooperative interactions with the canonical heterodimeric partner U2AF35 and the U2 snRNP com- polypyrimidine tract binding protein, U2AF65. Analy- plex to the splicing branch site with the initiation of the first sis of ␣CP-targeted exons predicts a substantial im- of two trans-esterification reactions (8). pact on fundamental cell functions. These findings Control of splice acceptor activity can be mediated by lead us to conclude that the ␣CPs play a direct and altering the efficiency and/or productivity of the interac- global role in modulating the splicing activity and in- tions between U2AF65 and the PPT (8). For example, the clusion of an array of cassette exons, thus driving RNA binding proteins PTB and hnRNPC have been pro- a novel pathway of splice site regulation within the posed to repress splice acceptor utilization by blocking the / mammalian transcriptome. binding and or activity of U2AF65 at the PPT (11,14,15). The mechanistic details of repression by these two fac-
*To whom correspondence should be addressed.Tel: +1 215 898 7834; Fax: +1 215 573 5892; Email: [email protected] Correspondence may also be addressed to Xinjun Ji. Tel: +1 215 898 4250; Fax: +1 215 573 5157; Email: [email protected] Correspondence may also be addressed to Yi Xing. Tel: +1 310 825 6806; Fax: +1 310 206 3663; Email: [email protected] †These authors contributed equally to this work as the first authors.