Project description: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 novel 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 25-50% decrease in transcription of both intron-containing and intron-less genes on a genomewide scale. 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 region of both intronic and non-intronic genes. These findings demonstrate that Prp19 has a novel role in the initiation step of transcription in budding yeast.
Project description:Small RNAs of the Piwi-interacting RNA class (piRNA) play a key role in controlling the activity of transposable elements (TEs) in the animal germ line. In some arthropod species, including mosquitoes, the vectors of malaria and other pathogens, the piRNA pathway is active not only in the gonads, where it controls TE activity, but also in somatic tissues, where its targets and functions are less clear. It is thought that the piRNA pathway in somatic tissues is involved in the antiviral response and the modulation of TE activity. Here, we studied the features of small RNA production in head and thorax somatic tissues of Anopheles coluzzii focusing on the small RNAs processed from protein-coding gene mRNAs. We revealed tissue and sex specificity in the production of small RNAs derived from the genic transcripts. Genic small RNAs of 24-30 nt in length are atypical RNAs that lack the hallmarks of piRNAs. The majority of these short RNAs are derived from mitochondrial and nuclear genes involved in energy metabolism. We discuss the peculiarities of the piRNA biogenesis in Anopheles species which may result in the production of genic small RNAs in the somatic tissues.