Gilmartin G M, Fleming E S, Oetjen J, Graveley B R
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405.
Genes Dev. 1995 Jan 1;9(1):72-83. doi: 10.1101/gad.9.1.72.
The endonucleolytic cleavage and polyadenylation of a pre-mRNA in mammalian cells requires two cis-acting elements, a highly conserved AAUAAA hexamer and an amorphous U- or GU-rich downstream element, that together constitute the "core" poly(A) site. The terminal redundancy of the HIV-1 pre-mRNA requires that the processing machinery disregard a core poly(A) site at the 5' end of the transcript, and efficiently utilize an identical signal that resides near the 3' end. Efficient processing at the 3' core poly(A) site, both in vivo and in vitro, has been shown to require sequences 76 nucleotides upstream of the AAUAAA hexamer. In this report we demonstrate that this HIV-1 upstream element interacts directly with the 160-kD subunit of CPSF (cleavage polyadenylation specificity factor), the factor responsible for the recognition of the AAUAAA hexamer. The presence of the upstream element in the context of the AAUAAA hexamer directs the stable binding of CPSF to the pre-mRNA and enhances the efficiency of poly(A) addition in reactions reconstituted with purified CPSF and recombinant poly(A) polymerase. Our results indicate that the dependence of HIV-1 3' processing on upstream sequences is a consequence of the suboptimal sequence context of the AAUAAA hexamer. We suggest that poly(A) site definition involves the recognition of multiple heterogeneous sequence elements in the context of the AAUAAA hexamer.
哺乳动物细胞中前体mRNA的核酸内切酶切割和聚腺苷酸化需要两个顺式作用元件，一个高度保守的AAUAAA六聚体和一个无定形的富含U或GU的下游元件，它们共同构成了“核心”聚腺苷酸化位点。HIV-1前体mRNA的末端冗余要求加工机制忽略转录本5'端的核心聚腺苷酸化位点，并有效利用位于3'端附近的相同信号。体内和体外实验均表明，3'核心聚腺苷酸化位点的有效加工需要AAUAAA六聚体上游76个核苷酸的序列。在本报告中，我们证明了这种HIV-1上游元件直接与CPSF（切割聚腺苷酸化特异性因子）的160-kD亚基相互作用，CPSF是负责识别AAUAAA六聚体的因子。在AAUAAA六聚体的背景下，上游元件的存在引导CPSF与前体mRNA稳定结合，并提高了用纯化的CPSF和重组聚腺苷酸聚合酶重构的反应中聚腺苷酸化的效率。我们的结果表明，HIV-1 3'加工对上游序列的依赖性是AAUAAA六聚体次优序列背景的结果。我们认为，聚腺苷酸化位点的定义涉及在AAUAAA六聚体背景下对多个异质序列元件的识别。
