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

哺乳动物物种中的许多基因经过交替的多聚腺苷酸化，导致转录本具有可变的3‘端。一些证据表明，Poly(A)位点的选择可能是发育调节的，也可能是以组织特异性的方式调节的，然而，负责调节替代PolyA位点选择的基本机制尚未阐明。在这项研究中，我证明了切割因子Im(CFIm)参与了3‘-非翻译区(UTR)内的PolyA位点的选择。CFIm是一种异源二聚体3‘末端加工复合体，在体外作用于Pre-mRNA上的其他加工因子。我从大肠杆菌提取液中制备了抗GST融合25 kDa CFIm亚基(CFIM25)的多克隆抗体。用RNAi方法将CFIm25在HeLa细胞中敲除，并用多克隆抗体进行Western blotting证实其下调水平。为了验证CFIm25是否参与了PolyA位点选择的调控，我用Northern blotting方法分析了TIMP-2、Syndecan 2、ERCC6和DHFR基因的替代PolyA位点选择。结果清楚地显示了CFIm25耗竭细胞中mRNAs分布的变化。此外，在TIMP2、Syndecan 2和ERCC6中的结果表明，在CFIm存在时，末端外显子最上游的Poly(A)位点主要不用于HeLa细胞，而在没有CFIm的情况下，最上游的Poly(A)位点占主导地位。因此，本研究提示CFIm在替代Poly(A)位点选择中的作用。在未来，本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一种新的基因治疗策略，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.