Beyer K, Dandekar T, Keller W
Department of Cell Biology, Biozentrum of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
J Biol Chem. 1997 Oct 17;272(42):26769-79. doi: 10.1074/jbc.272.42.26769.
Critical events in 3'-end processing of pre-mRNA are the recognition of the AAUAAA polyadenylation signal by cleavage and polyadenylation specificity factor (CPSF) and the binding of cleavage stimulation factor (CstF) via its 64-kDa subunit to the downstream element. The stability of this CPSF.CstF.RNA complex is thought to determine the efficiency of 3'-end processing. Since downstream elements reveal high sequence variability, in vitro selection experiments with highly purified CstF were performed to investigate the sequence requirements for CstF-RNA interaction. CstF was purified from calf thymus and from HeLa cells. Surprisingly, calf thymus CstF contained an additional, novel form of the 64-kDa subunit with a molecular mass of 70 kDa. RNA ligands selected by HeLa and calf thymus CstF contained three highly conserved sequence elements as follows: element 1 (AUGCGUUCCUCGUCC) and two closely related elements, element 2a (YGUGUYN0-4UUYAYUGYGU) and element 2b (UUGYUN0-4AUUUACU(U/G)N0-2YCU). All selected sequences tested functioned as downstream elements in 3'-end processing in vitro. A computer survey of the EMBL data library revealed significant homologies to all selected elements in naturally occurring 3'-untranslated regions. The majority of element 2a homologies was found downstream of coding sequences. Therefore, we postulate that this element represents a novel consensus sequence for downstream elements in 3'-end processing of pre-mRNA.
前体mRNA 3'末端加工过程中的关键事件包括切割和聚腺苷酸化特异性因子（CPSF）对AAUAAA聚腺苷酸化信号的识别，以及切割刺激因子（CstF）通过其64 kDa亚基与下游元件的结合。这种CPSF·CstF·RNA复合物的稳定性被认为决定了3'末端加工的效率。由于下游元件具有高度的序列变异性，因此进行了使用高度纯化的CstF的体外选择实验，以研究CstF与RNA相互作用的序列要求。CstF从小牛胸腺和HeLa细胞中纯化得到。令人惊讶的是，小牛胸腺CstF含有一种额外的、分子量为70 kDa的新型64 kDa亚基形式。由HeLa和小牛胸腺CstF选择的RNA配体包含三个高度保守的序列元件，如下所示：元件1（AUGCGUUCCUCGUCC）以及两个密切相关的元件，元件2a（YGUGUYN0 - 4UUYAYUGYGU）和元件2b（UUGYUN0 - 4AUUUACU(U/G)N0 - 2YCU）。所有测试的选定序列在体外3'末端加工中均作为下游元件发挥作用。对EMBL数据库的计算机检索显示，在天然存在的3'非翻译区中与所有选定元件具有显著同源性。元件2a的大多数同源性位于编码序列的下游。因此，我们推测该元件代表前体mRNA 3'末端加工中下游元件的一种新型共有序列。
