| Yhteenveto: | <p>Human small non-coding RNAs (snRNAs) are required for expression of the vast majority of our protein-coding genes due to their important role in pre-mRNA processing events. Transcription initiation of snRNA genes is mediated by transcription factors like Oct-1, which recognize the DSE element, and by PTF, which recognizes the PSE and nucleates a pre-initiation complex (PIC) comprising general transcription factors TFIIA, B, C, E, F and H, TBP and some TBPassociated factors, and Mediator. RNA polymerase II (pol II) is then recruited with the carboxylterminal domain (CTD) of the large subunit in a hypophosphorylated form. In humans, the CTD comprises 52 repeats of the consensus heptapeptide, Y<sub>1</sub>S<sub>2</sub>P<sub>3</sub>T<sub>4</sub>S<sub>5</sub>P<sub>6</sub>S<sub>7</sub>. After initiation, the pol II CTD is phosphorylated on S2, S5 and S7 as the polymerase progresses into elongation, which is mediated by the little elongation complex (LEC) and finally 3’ end formation and termination are mediated by recognition of the 3’ box RNA processing element by the Integrator complex. It has been shown that there is compulsory coupling between the promoter element PSE and the 3’ box; transcription by pol II must initiate from a PSE-containing promoter for the 3’ box to be recognised. However, the mechanism underlying this coupling is still unclear. In order to better understand how snRNA genes are regulated, I performed an unbiased dCas9-mediated pulldown approach to characterize the transcription machinery present on the DSE and PSE of the U2 snRNA gene. This analysis has identified some novel potential players in snRNA gene expression.</p> <p>After initiation of transcription of protein-coding genes, phosphorylation of S5 of the pol II CTD by the cyclin-dependent kinase (CDK) 7 subunit of TFIIH facilitates promoter escape and 5' capping of the emerging transcript. CTD S2 phosphorylation by the CDK9 subunit of the super elongation complex (SEC) and CDK12 facilitates processive elongation, splicing and recognition of the polyadenylation (poly(A)) signal in the nascent RNA. It has been shown that the activity of CDK9 is required for recognition of the snRNA gene 3' box. However, the roles of other CDKs was unknown. Characterisation of the in vivo roles of the CDKs has now been facilitated by using CRISPR/Cas9 genome engineering to produce cell lines with analog-sensitive (as) versions of many of these CDKs. In order to determine the roles of several CDKs in snRNA gene transcription, I performed pol II chromatin immunoprecipitation (ChIP) assays, RNase protection assays and immunoprecipitation/mass spectrometry analysis after specific inhibition of analog-sensitive CDKs. I have confirmed the role of CDK9 in expression of the human U2 snRNA gene and uncovered redundant roles for CDK12 and CDK13 in recognition of the 3' box.</p>
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