Coupling mRNA processing with transcription elongation

描述(申请人提供)：准确的前-信使核糖核酸的处理对于正确的基因表达和细胞的生存是必不可少的。在真核生物中，前mRNA的加工是以共转录的方式发生的，而连接这些过程的主要中介是RNA聚合酶II(PolII)的C末端结构域(CTD)，因为CTD的缺失抑制了前mRNA的加工。我们的实验室最近观察到，RNA聚合酶II活性部位的点突变也抑制了前mRNA的加工。物种间polII结构的保守性表明，这些突变可能会影响伸长速度，尽管这些突变在多大程度上改变伸长速度仍有待确定。转录延伸的速度如何调节前-mRNA的处理也还没有被解释。在本研究中，我建议通过以下方法来阐明polII催化核心的突变如何影响转录延伸和前mRNA加工的机制：1)使用染色质免疫沉淀(CHIP)和双分子荧光互补(BIFC)的组合是否会改变这些点突变体中加工因子的招募；2)使用无G盒的体外转录延伸试验，RNA聚合酶II活性部位的突变对延伸率的影响程度如何。我还将讨论3)核心突变是否会影响芯片使用检测Ser2或Ser5磷酸化的抗体进行CTD修饰，以及4)通过一系列包含核酶序列的构建来切断RNA和转录机制之间的联系，来确定mRNA转录本在加工因子招募中的作用。这项研究的结果将阐明转录延伸如何影响加工因子招募到激活的转录位点，并可能揭示转录和前mRNA加工之间的新联系。 相关性：转录延长的解除调控与人类疾病有关，如急性髓系白血病、Cockayne综合征和Von Hippel-Lindau病，尽管转录延长改变导致疾病的机制尚不清楚。虽然异常的转录延伸率会改变转录本的总数，但我们的实验室有证据表明，转录延伸率的改变也可能影响Pre-mRNA的处理。因此，我们的研究可能有助于阐明转录延伸率改变是如何导致人类疾病中基因表达异常的。

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.