TY - JOUR TI - Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing AU - Wongpalee, Somsakul Pop AU - Vashisht, Ajay AU - Sharma, Shalini AU - Chui, Darryl AU - Wohlschlegel, James A AU - Black, Douglas L A2 - Nilsen, Timothy W VL - 5 PY - 2016 DA - 2016/11/24 SP - e19743 C1 - eLife 2016;5:e19743 DO - 10.7554/eLife.19743 UR - https://doi.org/10.7554/eLife.19743 AB - 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. KW - RNA binding protein KW - splicing KW - gene regulation KW - ribonucleoprotein JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -