Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing.

Somsakul Pop Wongpalee,James A Wohlschlegel,Shalini Sharma,Ajay Vashisht,Darryl Chui,Douglas L Black

doi:10.7554/elife.19743

Somsakul Pop Wongpalee, James A Wohlschlegel + Show 4 more

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https://doi.org/10.7554/elife.19743

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Abstract

Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition. The active early complex formed in the absence of PTBP1 proceeded to assemble an EDC with the eviction of hnRNP proteins, the late recruitment of SR proteins, and binding of the U2 snRNP. These results demonstrate that during early stages of splicing, exon RNP complexes are highly dynamic with many proteins failing to bind during PTBP1 arrest.

Highlights

Alternative pre-mRNA splicing is a key regulatory process that determines the output of most genes of higher eukaryotes (Pan et al, 2008; Lee and Rio, 2015; Merkin et al, 2012; BarbosaMorais et al, 2012; Fu and Ares, 2014)
We found that Src alternative exon N1 failed to properly interact with the active exon definition complex (EDC) assembled onto the constitutive exon 4 downstream, despite the presence of the U1 snRNP bound to the 5’ splice site of the N1 exon (Sharma et al, 2008)
We found that PTBP1 binding allows normal U2AF65 and U1 snRNP recruitment to this exon, but prevents U2 snRNP assembly and EDC formation

Summary

Introduction

Alternative pre-mRNA splicing is a key regulatory process that determines the output of most genes of higher eukaryotes (Pan et al, 2008; Lee and Rio, 2015; Merkin et al, 2012; BarbosaMorais et al, 2012; Fu and Ares, 2014). Splicing choices are directed by proteins of multiple classes and structural families that bind to the nascent pre-mRNA to alter spliceosome assembly (Lee and Rio, 2015; Fu and Ares, 2014). Other proteins are more generally classed as the heterogeneous nuclear ribonucleoproteins (hnRNP proteins) that belong to multiple structural families and bind to specific sites throughout the nascent pre-mRNA (Busch and Hertel, 2012; Dreyfuss et al, 1993). Depending on the family and binding site location, hnRNP proteins can act to either repress or stimulate splicing of particular exons or splice sites (Fu and Ares, 2014) The interactions of these pre-mRNA-bound regulatory proteins with the general splicing apparatus or spliceosome are largely unknown

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