Coupling mRNA processing with transcription elongation

描述（由申请人提供）：准确处理前mrna对于正确的基因表达和细胞存活至关重要。在真核生物中，mrna前加工是共转录发生的，耦合这些过程的主要介质是RNA聚合酶II （pol II）的c端结构域（CTD），因为CTD的缺失抑制了mrna前加工。我们的实验室最近观察到RNA聚合酶II活性位点的点突变也抑制了前mrna的加工。物种间pol II结构的保守性表明，这些突变可能影响延伸速度，但这些突变在多大程度上改变延伸率仍有待确定。转录延伸率如何调节pre-mRNA加工也仍未解释。在这项研究中，我建议阐明pol II催化核心突变如何影响转录延伸和前mrna加工的机制，通过解决以下问题：1)使用染色质免疫沉淀（ChIP）和双分子荧光互补（BiFC）的组合是否改变这些点突变体中加工因子的募集；2)使用体外转录延伸，RNA聚合酶II活性位点突变对延伸率的影响程度使用无g磁带进行分析。我还将讨论3)核心突变是否会影响检测Ser2或Ser5磷酸化的ChIP修饰CTD； 4)使用一系列含有核酶序列的构建体来切断RNA与转录机制之间的联系，确定mRNA转录物在招募加工因子中的作用。这项研究的结果将阐明转录伸长如何影响加工因子招募到活性转录位点，并可能揭示转录和mrna前加工之间的新联系。

DESCRIPTION (provided by applicant): Accurate processing of pre-mRNA is essential in proper gene expression and therefore, survival of a cell. In eukaryotes, pre-mRNA processing occurs co-transcriptionally and the major mediator in coupling these processes is the C-terminal domain (CTD) of RNA polymerase II (pol II) as CTD deletion inhibits pre-mRNA processing. Our lab has recently observed that point mutations in RNA polymerase II active site also inhibit pre-mRNA processing. Conservation of the pol II structure between species suggests that these mutations could affect the elongation speed although to what extent these mutations alter elongation rate remains to be determined. How the rate of transcription elongation regulate pre-mRNA processing also remains unexplained. In this study, I propose to elucidate the mechanism behind how mutations at the catalytic core of pol II affect transcription elongation and pre-mRNA processing by addressing 1) whether the recruitment of processing factors is altered in these point mutants using a combination of chromatin immunoprecipitation (ChIP) and bimolecular fluorescence complementation (BiFC) 2)to what degree the elongation rate is affected by the mutations in the RNA polymerase II active site using an in vitro transcription elongation assay using G-less cassettes. I will also address 3) whether mutation at the core effects CTD modification by ChIP with antibodies that detect Ser2 or Ser5 phosphorylation and 4) determine the role of mRNA transcript in recruitment of processing factors using series of constructs containing ribozyme sequences to sever the ties between RNA and the transcription machinery. Results from this study will elucidate how transcription elongation influences processing factor recruitment to the active transcription site and may reveal a new connection between transcription and pre-mRNA processing. Relevance: Deregulation of transcription elongation is implicated in human diseases, such as in acute myeloid leukemia, Cockayne syndrome, and Von Hippel-Lindau disease, although the mechanism behind how altered transcription elongation contributes to disease remains unclear. While abnormal transcription elongation rate can change the overall number of transcripts, our lab has evidence that the altered transcription elongation rate may also affect processing of pre-mRNA. Therefore, our study may shed a light onto how altered transcription elongation rate contributes to abnormal gene expression in human diseases.