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

在哺乳动物物种中，许多基因经历了选择性聚腺苷酸化，导致转录本具有可变的3'端。一些证据表明，聚(A)位点选择可能是发育或以组织特定方式调节的，然而，负责调节选择性聚(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poly(A)的位点选择。CFIm是一种异二聚体的3'端加工复合物，其功能是在体外将其他加工因子组装在pre-mRNA上。从大肠杆菌提取物中纯化CFIm的25 kDa亚基（cfi25），制备抗GST多克隆抗体。采用RNAi法在HeLa细胞中敲低CFIm25，用多克隆抗体进行western blotting检测敲低水平。为了研究CFIm25是否参与poly(A)位点选择的调控，我利用northern blotting分析了TIMP-2、syndecan 2、ERCC6和DHFR基因的备选poly(A)位点选择。结果清楚地显示了在CFIm25缺失的细胞中mrna分布的变化。此外，TIMP2， syndecan 2和ERCC6的结果表明，当CFIm存在时，末端外显子最上游的poly(A)位点在HeLa细胞中不主要使用，而在不存在CFIm时，最上游的poly(A)位点占主导地位。因此，本研究提示CFIm在选择性聚(a)位点选择中的作用。在未来，该研究可能通过在分子水平上理解聚(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。

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.