Graveley B R, Fleming E S, Gilmartin G M
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, Stafford Hall, University of Vermont, Burlington, Vermont 05405, USA.
J Biol Chem. 1996 Dec 27;271(52):33654-63. doi: 10.1074/jbc.271.52.33654.
Efficient cleavage and polyadenylation at the human immunodeficiency virus type-1 (HIV-1) poly(A) site requires an upstream 3'-processing enhancer to overcome the suboptimal sequence context of the AAUAAA hexamer. The HIV-1 3'-processing enhancer functions to stabilize the association of the pre-mRNA with cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of the AAUAAA hexamer. Intriguingly, in the absence of the 3'-processing enhancer, CPSF binding and polyadenylation efficiency could be restored to near wild-type levels upon replacement of the 14-nucleotide region immediately 5' of the HIV-1 AAUAAA hexamer (the B segment) by the analogous sequences from the efficient adenovirus L3 poly(A) site. To further investigate the contributions of RNA sequence and structure to poly(A) site recognition, we have used an in vitro selection system to identify B segment sequences that enhance the polyadenylation efficiency of a pre-cleaved RNA lacking a 3'-processing enhancer. The final RNA selection pool was composed of two predominant classes of RNAs. Nuclease probing revealed that the selected sequences restored an RNA conformation that facilitates recognition of the AAUAAA hexamer by CPSF. These results indicate that both the sequence and structural context of the AAUAAA hexamer contribute to poly(A) site recognition by CPSF.
在人类免疫缺陷病毒1型（HIV-1）聚腺苷酸化位点进行高效切割和聚腺苷酸化需要一个上游3'加工增强子，以克服AAUAAA六聚体次优的序列背景。HIV-1 3'加工增强子的作用是稳定前体mRNA与切割和聚腺苷酸化特异性因子（CPSF）的结合，CPSF是负责识别AAUAAA六聚体的因子。有趣的是，在没有3'加工增强子的情况下，将HIV-1 AAUAAA六聚体（B段）5'端紧邻的14个核苷酸区域替换为高效腺病毒L3聚腺苷酸化位点的类似序列后，CPSF结合和聚腺苷酸化效率可恢复到接近野生型水平。为了进一步研究RNA序列和结构对聚腺苷酸化位点识别的贡献，我们使用了一种体外筛选系统来鉴定能够增强缺乏3'加工增强子的预切割RNA聚腺苷酸化效率的B段序列。最终的RNA筛选文库由两类主要的RNA组成。核酸酶探测显示，所选序列恢复了一种有利于CPSF识别AAUAAA六聚体的RNA构象。这些结果表明，AAUAAA六聚体的序列和结构背景都有助于CPSF对聚腺苷酸化位点的识别。
