Human pre-mRNA cleavage factor IIm contains homologs of yeast proteins and bridges two other cleavage factors
Human pre-mRNA cleavage factor IIm contains homologs of yeast proteins and bridges two other cleavage factors
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DOI：
10.1093/emboj/19.21.5895
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发表时间：
2000-11-01
期刊：
影响因子：
11.4
通讯作者：
Keller, W
中科院分区：
文献类型：
--
作者：
de Vries, H;Rüegsegger, U;Keller, W
哺乳动物Pre-mRNAs在体外需要6种不同的蛋白质因子，通过内切切割和多聚腺苷作用形成3‘端。其中5个因子已被纯化并克隆了其大部分组分，但切割因子IIM(CFIIM)仍未被鉴定。我们通过几个层析步骤从HeLa细胞核提取液中纯化了CFIIM。在纯化过程中，CFIIM活性分为两个组分，一个是切割反应所必需的(CF IIA(M))，另一个是促进切割反应的(CF IIBm)。Cf IIA(M)组分含有两个酵母3‘末端加工因子Pcf11p和Clp1p的人类同源物，以及切割因子I-m(CFi-m)和几种剪接和转录因子。我们报道了hClp1的克隆，并证明它是CFIIA(M)的一个真正的亚基。针对hClp1的抗体会耗尽HeLa细胞核提取液的切割活性，但不会耗尽多聚腺苷化活性。HClp1与CFi-m以及切割和多聚腺苷化特异性因子CPSF相互作用，表明它在切割复合体中连接了这两个3‘端加工因子。
Six different protein factors are required in vitro for 3' end formation of mammalian pre-mRNAs by endonucleolytic cleavage and polyadenylation. Five of the factors have been purified and most of their components cloned, but cleavage factor IIm (CF IIm) remained uncharacterized. We have purified CF IIm from HeLa cell nuclear extract by several chromatographic steps. During purification, CF IIm activity separated into two components, one essential (CF IIA(m)) and one stimulatory (CF IIBm) for the cleavage reaction. CF IIA(m) fractions contain the human homologs of two yeast 3' end processing factors, Pcf11p and Clp1p, as well as cleavage factor I-m (CF I-m) and several splicing and transcription factors. We report the cloning of hClp1 and show that it is a genuine subunit of CF IIA(m). Antibodies directed against hClp1 deplete cleavage activity, but not polyadenylation activity from HeLa cell nuclear extract. hClp1 interacts with CF I-m and the cleavage and polyadenylation specificity factor CPSF, suggesting that it bridges these two 3' end processing factors within the cleavage complex.