de Vries H, Rüegsegger U, Hübner W, Friedlein A, Langen H, Keller W
Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Germany.
EMBO J. 2000 Nov 1;19(21):5895-904. doi: 10.1093/emboj/19.21.5895.
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 II(m) (CF II(m)) remained uncharacterized. We have purified CF II(m) from HeLa cell nuclear extract by several chromatographic steps. During purification, CF II(m) activity separated into two components, one essential (CF IIA(m)) and one stimulatory (CF IIB(m)) 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.
在体外，哺乳动物前体mRNA通过核酸内切酶切割和聚腺苷酸化形成3'末端需要六种不同的蛋白质因子。其中五种因子已被纯化，并且它们的大多数组分已被克隆，但切割因子II（m）（CF II（m））仍未被鉴定。我们通过几个色谱步骤从HeLa细胞核提取物中纯化了CF II（m）。在纯化过程中，CF II（m）活性分离为两个组分，一个对切割反应是必需的（CF IIA（m）），另一个是刺激性的（CF IIB（m））。CF IIA（m）级分包含两种酵母3'末端加工因子Pcf11p和Clp1p的人类同源物，以及切割因子I（m）（CF I（m））和几种剪接和转录因子。我们报道了hClp1的克隆，并表明它是CF IIA（m）的一个真正亚基。针对hClp1的抗体耗尽了HeLa细胞核提取物中的切割活性，但没有耗尽聚腺苷酸化活性。hClp1与CF I（m）以及切割和聚腺苷酸化特异性因子CPSF相互作用，表明它在切割复合物中桥接这两个3'末端加工因子。
