Coupling mRNA processing with transcription elongation

将 mRNA 加工与转录延伸相结合

• 批准号: 7220146
• 负责人: SOOJIN KIM
• 金额: $ 3.35万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-02 至 2007-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7220146
• 关键词: Active Sites; Acute Myelocytic Leukemia; Address; Affect; Amino Acid Substitution; Antibodies; Binding; Biological Assay; C-terminal; Catalytic Domain; Catalytic RNA; Cells; Cockayne Syndrome; Coupled; Coupling; DNA-Directed RNA Polymerase; Disease; Eukaryota; Eukaryotic Cell; Fluorescence; Gene Expression; Genetic Transcription; Heterogeneous Nuclear RNA; In Vitro; Kinetics; Light; Link; Mediator of activation protein; Messenger RNA; Modification; Molecular; Mutation; Numbers; Phosphorylation; Play; Point Mutation; Polyadenylation; Polymerase; Process; RNA; RNA Polymerase II; RNA Splicing; RNA polymerase II largest subunit; Rate; Recruitment Activity; Role; Series; Site; Speed; Structure; Transcript; Transcription Elongation; Transcription Process; Von Hippel-Lindau Syndrome; base; chromatin immunoprecipitation; human disease; mRNA Precursor; mutant

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.

描述（由申请人提供）：前mRNA的准确加工对于基因的正确表达以及细胞的生存至关重要。在真核生物中，前mRNA加工共转录发生，并且耦合这些过程的主要介质是RNA聚合酶II（pol II）的C末端结构域（CTD），因为CTD缺失抑制前mRNA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。物种之间的pol II结构的保守性表明，这些突变可能会影响延伸速度，尽管这些突变在多大程度上改变延伸率仍有待确定。转录延长的速率如何调节前mRNA的加工也仍然没有解释。在本研究中，我建议阐明pol II催化核心的突变如何影响转录延伸和前体mRNA加工的机制，方法是：1）使用染色质免疫沉淀（ChIP）和双分子荧光互补（BiFC）2）的组合，在这些点突变体中是否改变了加工因子的募集。使用无G盒的体外转录延伸测定，RNA聚合酶II活性位点中的突变对延伸速率的影响程度。我还将解决3）在核心突变是否影响CTD的修改，通过ChIP与抗体检测Ser 2或Ser 5磷酸化和4）确定mRNA转录的作用，在招聘的加工因子使用一系列的构建体含有核酶序列切断RNA和转录机制之间的联系。这项研究的结果将阐明转录延长如何影响加工因子的招聘到活性转录位点，并可能揭示转录和前mRNA加工之间的新联系。 相关性：转录延长的失调与人类疾病有关，例如急性髓性白血病、Cockayne综合征和Von Hippel-Lindau病，尽管转录延长改变如何导致疾病的机制仍不清楚。虽然异常的转录延伸率可以改变转录物的总数，但我们实验室有证据表明，转录延伸率的改变也可能影响前mRNA的加工。因此，我们的研究可能会揭示如何改变转录延伸率有助于人类疾病中的异常基因表达。