Abstract
We have previously demonstrated that the transcription-dependent interaction of the promoter and terminator ends of a gene, which results in the formation of a gene loop, is facilitated by the interaction of the general transcription factor TFIIB with the CF1, CPF and Rat1 termination complexes. To further elucidate the protein-protein interactions that stabilize gene loop, we performed mass spectrometry of affinity purified termination complexes from chromatin fraction. Quantitative proteomic analysis revealed additional interactions of termination factors with TFIID and SAGA complex. Since gene looping of intron-containing genes involves additional contacts of the promoter and terminator with the intron, we examined if termination factors interact with the splicing factors as well. All three termination complexes displayed statistically significant interactions with Prp19, Prp43, Sub2, Snu114, Brr2 and Smb1 splicing factors. Since Prp43 and Prp19 consistently emerged as the interactor of both initiation and termination factors, we affinity-purified both and performed mass spectrometry. Prp19 exhibited interactions with subunits of TFIID, CPF complex, and the RSC chromatin remodeling complex. These interactions were observed exclusively in the chromatin context, thereby implicating the factor in transcription of protein coding genes. Since fewer than 4% of yeast genes contain introns, we hypothesized that Prp19 might have a broader role in RNAPII transcription cycle. Auxin-mediated depletion of Prp19 resulted in about two-fold decrease in transcription of a subset of both intron-containing and intron-lacking genes. Specifically, the promoter recruitment of TBP registered a significant decline in the absence of Prp19. Chromatin immunoprecipitation (ChIP) analysis revealed crosslinking of Prp19 to the promoter proximal as well as downstream regions of both intronic and non-intronic genes. These findings demonstrate that Prp19 has a novel role in the initiation step of transcription in yeast.