Molecular Assembly of Exon Definition Complex and Its Regulation for Alternative Precursor-messenger RNA Splicing by Polypyrimidine-tract Binding Protein PTBP1

Alternative pre-mRNA splicing (AS) plays a major role in gene expression in higher eukaryotes, especially in vertebrates. Regulation of AS is primarily accomplished by splicing factors that bind to regulatory elements on pre-mRNAs and influence spliceosomal complex assembly. One important splicing factor is the polypyrimidine tract-binding protein 1 (PTBP1), well known for its repressor activity. In repressing an alternative exon, PTBP1 uses different mechanisms depending on the location of its binding sites. PTBP1 can compete with U2AF65 to bind the polypyrimidine tract at the 3’ splice site if it contains the PTBP1 binding sites. Alternatively, when PTBP1 is bound to an exon, it can block the recruitment of U2AF65 across an exon or the cross-intron interactions to an adjacent exon. However, PTBP1 binding sites are often located further within both introns flanking an alternative exon. The mechanism behind this type of alternative exon repression is poorly characterized. Therefore, we used a modified version of an alternative N1 exon of c-src pre-mRNA to characterize this type of repression mechanism. We found that PTBP1 stalls an exon in an early exon complex, inhibiting its transition to an exon definition complex (EDC), which is required for subsequent spliceosomal complex assembly. Using quantitative mass spectrometry to analyze PTBP1-repressed early exon complex, de-repressed early exon complex and the EDC, we identified that PTBP1 accomplishes this, in part, by blocking the association of two ATP-dependent RNA helicases—DDX5 and DDX17, which we found playing an important role promoting efficient formation of the EDC in vitro and exon inclusion in vivo. Interestingly, during the normal transition of the exon from early exon complex to the EDC, we detected the removal of hnRNP proteins and substantial recruitment of SR proteins, concurrent with U2 snRNP recruitment. Not only did these data reveal for the first time the assembly of the EDC, but also suggest that there are remodeling events required prior to the exon definition process. We believe that these remodeling events could be attributed to actions of ATP-dependent RNA helicases DDX5 and DDX17.