THE 64-KILODALTON SUBUNIT OF THE CSTF POLYADENYLATION FACTOR BINDS TO PRE-MESSENGER-RNAS DOWNSTREAM OF THE CLEAVAGE SITE AND INFLUENCES CLEAVAGE SITE LOCATION
THE 64-KILODALTON SUBUNIT OF THE CSTF POLYADENYLATION FACTOR BINDS TO PRE-MESSENGER-RNAS DOWNSTREAM OF THE CLEAVAGE SITE AND INFLUENCES CLEAVAGE SITE LOCATION
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DOI：
10.1128/mcb.14.10.6647
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发表时间：
1994-10-01
影响因子：
5.3
通讯作者：
SHENK, T
中科院分区：
文献类型：
--
作者：
MACDONALD, CC;WILUSZ, J;SHENK, T
CstF聚腺苷酸化因子是前体mRNA的有效切割和聚腺苷酸化所需的多亚基复合物。使用RNase H介导的映射技术，我们表明，64 kDa的CstF亚基可以光交联前mRNA在富含U的区域位于下游的猿猴病毒40晚和腺病毒L3前mRNA的切割位点。交联的这种位置特异性是CstF与聚腺苷酸化复合物相互作用的结果，因为64-kDa蛋白本身在前mRNA模板上的多个位置处交联。在多聚腺苷酸化过程中，四个连续的U残基可以取代猿猴病毒40前体mRNA上的天然下游富含U的序列，从而在下游位置介导有效的64-kDa蛋白质交联。此外，U段的位置不仅使64-kDa多肽能够与前mRNA交联，而且还影响切割位点。GenBank数据库的搜索显示，哺乳动物的多聚腺苷酸化位点的相当大的一部分进行了四个或更多个连续的U残基的位置，使他们应该作为网站的相互作用与64 kDa的蛋白质下游的切割位点。我们的研究结果表明，多聚腺苷酸化机械物理上跨越切割位点，引导切割因子之间的位置位于上游AAUAAA基序，切割和多聚腺苷酸化特异性因子被认为是相互作用，和下游U-丰富的结合位点的64 kDa亚基的CstF。
The CstF polyadenylation factor is a multisubunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. Using an RNase H-mediated mapping technique, we show that the 64-kDa subunit of CstF can be photo cross-linked to pre-mRNAs at U-rich regions located downstream of the cleavage site of the simian virus 40 late and adenovirus L3 pre-mRNAs. This positional specificity of cross-linking is a consequence of CstF interaction with the polyadenylation complex, since the 64-kDa protein by itself is cross-linked at multiple positions on a pre-mRNA template. During polyadenylation, four consecutive U residues can substitute for the native downstream U-rich sequence on the simian virus 40 pre-mRNA, mediating efficient 64-kDa protein cross-linking at the downstream position. Furthermore, the position of the U stretch not only enables the 64-kDa polypeptide to be cross-linked ts the pre-mRNA but also influences the site of cleavage. A search of the GenBank database revealed that a substantial portion of mammalian polyadenylation sites carried four or more consecutive U residues positioned so that they should function as sites for interaction with the 64-kDa protein downstream of the cleavage site. Our results indicate that the polyadenylation machinery physically spans the cleavage site, directing cleavage factors to a position located between the upstream AAUAAA motif, where the cleavage and polyadenylation specificity factor is thought to interact, and the downstream U-rich binding site for the 64-kDa subunit of CstF.