Opposing Effects of Inhibiting Cap Addition and Cap Methylation on Polyadenylation during Vesicular Stomatitis Virus mRNA Synthesis
Opposing Effects of Inhibiting Cap Addition and Cap Methylation on Polyadenylation during Vesicular Stomatitis Virus mRNA Synthesis
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DOI：
10.1128/jvi.02162-08
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发表时间：
2009-02-15
影响因子：
5.4
通讯作者：
Whelan, Sean P. J.
中科院分区：
文献类型：
--
作者：
Li, Jianrong;Rahmeh, Amal;Whelan, Sean P. J.
水疱性口炎病毒（VSV）的多功能大(L)聚合酶蛋白具有RNA合成所必需的酶活性，包括mRNA帽添加和聚腺苷化。我们之前绘制了氨基酸残基G1154、T1157、H1227和R1228，这些氨基酸残基存在于L的保守区V （CRV）中，是mRNA cap添加所必需的。在这里，我们表明丙氨酸取代这些残基也影响3'端形成。具体来说，帽缺陷聚合酶产生的截断转录本在其3'端含有富含a的序列，主要终止于N基因的前500个核苷酸（nt）内。为了研究帽缺陷聚合酶如何对存在于每个基因连接处的真正VSV终止和重新启动信号作出反应，我们使用包含插入(I)在先导- n基因连接处的基因的模板重建了RNA合成。I基因的大小在382 ~ 1098 nt之间，通常转录成全长无帽转录本。除了缺乏帽结构外，由帽缺陷聚合酶合成的全长I转录本缺乏真正的聚腺苷酸尾部，而含有0至24个a残基。此外，帽缺陷聚合酶也不能有效地复制下游基因。因此，L蛋白CRV中的单氨基酸替换抑制了cap加成，也抑制了基因组的聚腺苷化和序列转录。相反，L蛋白CRVI中的氨基酸替换K1651A完全抑制帽甲基化，导致mRNA的高聚腺苷化。这项研究表明，抑制帽帽添加和帽帽甲基化对VSV mRNA合成过程中的聚腺苷化有相反的影响，并提供证据支持正确的5‘帽形成与3’聚腺苷化之间的联系。
The multifunctional large (L) polymerase protein of vesicular stomatitis virus (VSV) contains enzymatic activities essential for RNA synthesis, including mRNA cap addition and polyadenylation. We previously mapped amino acid residues G1154, T1157, H1227, and R1228, present within conserved region V (CRV) of L, as essential for mRNA cap addition. Here we show that alanine substitutions to these residues also affect 3'-end formation. Specifically, the cap-defective polymerases produced truncated transcripts that contained A-rich sequences at their 3' termini and predominantly terminated within the first 500 nucleotides (nt) of the N gene. To examine how the cap-defective polymerases respond to an authentic VSV termination and reinitiation signal present at each gene junction, we reconstituted RNA synthesis using templates that contained genes inserted (I) at the leader-N gene junction. The I genes ranged in size from 382 to 1,098 nt and were typically transcribed into full-length uncapped transcripts. In addition to lacking a cap structure, the full-length I transcripts synthesized by the cap-defective polymerases lacked an authentic polyadenylate tail and instead contained 0 to 24 A residues. Moreover, the cap-defective polymerases were also unable to copy efficiently the downstream gene. Thus, single amino acid substitutions in CRV of L protein that inhibit cap addition also inhibit polyadenylation and sequential transcription of the genome. In contrast, an amino acid substitution, K1651A, in CRVI of L protein that completely inhibits cap methylation results in the hyperpolyadenylation of mRNA. This work reveals that inhibiting cap addition and cap methylation have opposing effects on polyadenylation during VSV mRNA synthesis and provides evidence in support of a link between correct 5' cap formation and 3' polyadenylation.