权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Functional analysis of CF-I complex regulating mRNA 3' cleavage

CF-I复合物调节mRNA 3切割的功能分析

基本信息

批准号：
18570159
负责人：
WADA Tadashi
金额：
$ 2.5万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570159/
关键词：
transcription poly(A) site mRNA RNA cleavage antibody UTR western blotting northern blotting 組換えタンパク質免疫沈降 RNA干渉組織特異的

项目摘要

Numerous genes in mammalian species undergo alternative polyadenylation, which results in transcripts with variable 3' end. Several lines of evidence suggest that poly(A) sites selection may be modulated developmentally or in a tissue specific manner, however, the fundamental mechanisms responsible for regulating alternative poly(A) site selection have not been elucidated. In this study, I showed involvement of cleavage factor Im (CFIm) in poly(A) site selection within a 3'- untranslated region (UTR). CFIm is a heterodimeric 3' end processing complex, which functions to assemble other processing factors on pre-mRNA in vitro. I made polyclonal antibodies against GST fused 25 kDa subunit of CFIm (CFIm25) purified from E. coli extracts. CFIm25 was knocked down in HeLa cells by RNAi method and the knock down level was confirmed by western blotting by using the polyclonal antibody. To examine whether CFIm25 is involved in the regulation of poly(A) sites selection, I analyzed alternative poly(A) site selection of TIMP-2, syndecan 2, ERCC6 and DHFR genes by northern blotting. Results clearly showed changes in the distribution of mRNAs in CFIm25 depleted cells. Moreover, results in TIMP2, syndecan 2 and ERCC6 indicate that in the presence of CFIm the most upstream poly(A) sites in the terminal exons are not primarily used in HeLa cells, while in the absence of CFIm the most upstream poly(A) sites predominate. Therefore, this study suggests a role for CFIm in alternative poly(A) site selection. In the future, this study may support a novel gene therapy strategy by understanding mechanisms of poly(A) sites selection at a molecular level and contribute to basic science such as developmental biology and cell biology.

哺乳动物中的许多基因都经历了交替的多聚腺苷酸化，这导致了3'端可变的转录本。一些证据表明，poly（A）位点选择可能在发育过程中或以组织特异性方式进行调节，然而，负责调节替代poly（A）位点选择的基本机制尚未阐明。在这项研究中，我发现切割因子Im（CFIm）参与了3 '-非翻译区（UTR）内的poly（A）位点选择。CFIm是一种异二聚体3'端加工复合物，其功能是在体外将其他加工因子组装在前体mRNA上。制备了抗CFIm 25亚基（CFIm 25）的多克隆抗体。大肠杆菌提取物。通过RNAi方法在HeLa细胞中敲低CFIm 25，并使用多克隆抗体通过Western印迹证实敲低水平。为了检测CFIm 25是否参与poly（A）位点选择的调节，我通过北方印迹分析了TIMP-2、syndecan 2、ERCC 6和DHFR基因的备选poly（A）位点选择。结果清楚地显示CFIm 25耗尽细胞中mRNA分布的变化。此外，TIMP 2、多配体蛋白聚糖2和ERCC 6的结果表明，在存在CFIm的情况下，末端外显子中最上游的poly（A）位点在HeLa细胞中不主要使用，而在不存在CFIm的情况下，最上游的poly（A）位点占主导地位。因此，该研究表明CFIm在替代性聚（A）位点选择中的作用。在未来，这项研究可能通过在分子水平上理解poly（A）位点选择的机制来支持新的基因治疗策略，并为发育生物学和细胞生物学等基础科学做出贡献。