GDP polyribonucleotidyltransferase domain of vesicular stomatitis virus polymerase regulates leader-promoter escape and polyadenylation-coupled termination during stop-start transcription.
GDP polyribonucleotidyltransferase domain of vesicular stomatitis virus polymerase regulates leader-promoter escape and polyadenylation-coupled termination during stop-start transcription.
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DOI：
10.1371/journal.ppat.1010287
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发表时间：
2022-03
期刊：
影响因子：
6.7
通讯作者：
Ogino T
中科院分区：
文献类型：
--
作者：
Ogino M;Green TJ;Ogino T
水泡性口炎病毒（VSV）L蛋白的非常规mRNA加帽酶（GDP多核糖核苷酸转移酶，PRNTase）结构域具有双功能“引发-加帽环”，其在独特的停止-开始转录循环期间控制前导RNA合成的末端从头起始和单顺反子mRNA的加帽。在这里，我们研究了螺旋结构上的碱性氨基酸残基在病毒RNA合成中直接连接到引发-加帽环上的作用，并确定了在停止-启动转录的不同步骤中引起先前未报告的缺陷表型的单点突变。残基R1183（R1183 A和R1183 K）的突变通过阻碍早期延伸而显著降低前导RNA合成活性，但不阻碍末端从头起始或生产性延伸，表明突变负面影响前导启动子的逃逸。另一方面，残基R1178（R1178 A和R1178 K）的突变降低了多腺苷酸化偶联终止基因连接处mRNA合成的效率，但不终止前导RNA合成的前导-N-基因连接处，导致产生更大量的异常多顺反子mRNA。相反，R1183和R1178残基对于帽形成活性不是必需的。R1183 K突变对VSV是致命的，而R1178 K突变使VSV减毒并触发感染细胞中多顺反子mRNA的产生。这些观察结果表明，PRNTase结构域在进行精确的停止-开始转录中发挥多种作用，超出了其在前mRNA加帽中的已知作用。水泡性口炎病毒（VSV），一种与狂犬病病毒密切相关的动物弹状病毒，已经作为理解弹状病毒（例如，狂犬病）和其他非节段负链（NNS）RNA病毒，如麻疹和埃博拉病毒。NNS RNA病毒聚合酶通过终止-启动转录机制，分别从其基因组的3′-末端前导区和内部基因顺序合成非编码前导RNA和单顺反子mRNA。NNS RNA病毒聚合酶的一个标志是存在一个独特的酶结构域，称为GDP多核糖核苷酸转移酶（PRNTase），它催化前体mRNA 5′-加帽，这是一种必需的mRNA修饰。我们最近的研究表明，VSV PRNTase结构域在基因组的3′-末端指导转录起始，以及在停止-启动转录过程中用双功能引发-加帽环指导前mRNA加帽。在这里，我们进一步表明，螺旋结构两侧的引物加帽环调节不仅在早期阶段的前导RNA合成的转录延长，但也多腺苷酸化耦合的转录终止在基因连接。这些发现表明，PRNTase结构域作为一个关键的调控结构域的停止-开始转录，以及前mRNA加帽的催化结构域。
The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase) domain of the vesicular stomatitis virus (VSV) L protein possesses a dual-functional "priming-capping loop" that governs terminal de novo initiation for leader RNA synthesis and capping of monocistronic mRNAs during the unique stop-start transcription cycle. Here, we investigated the roles of basic amino acid residues on a helix structure directly connected to the priming-capping loop in viral RNA synthesis and identified single point mutations that cause previously unreported defective phenotypes at different steps of stop-start transcription. Mutations of residue R1183 (R1183A and R1183K) dramatically reduced the leader RNA synthesis activity by hampering early elongation, but not terminal de novo initiation or productive elongation, suggesting that the mutations negatively affect escape from the leader promoter. On the other hand, mutations of residue R1178 (R1178A and R1178K) decreased the efficiency of polyadenylation-coupled termination of mRNA synthesis at the gene junctions, but not termination of leader RNA synthesis at the leader-to-N-gene junction, resulting in the generation of larger amounts of aberrant polycistronic mRNAs. In contrast, both the R1183 and R1178 residues are not essential for cap-forming activities. The R1183K mutation was lethal to VSV, whereas the R1178K mutation attenuated VSV and triggered the production of the polycistronic mRNAs in infected cells. These observations suggest that the PRNTase domain plays multiple roles in conducting accurate stop-start transcription beyond its known role in pre-mRNA capping. Vesicular stomatitis virus (VSV), an animal rhabdovirus closely related to rabies virus, has served as a paradigm for understanding the basic molecular mechanisms of transcription and replication by rhabdoviruses (e.g., rabies) and other non-segmented negative strand (NNS) RNA viruses, such as measles and Ebola. NNS RNA viral polymerases sequentially synthesize the non-coding leader RNA and monocistronic mRNAs from the 3′-terminal leader region and internal genes, respectively, on their genomes by the stop-start transcription mechanism. A hallmark of NNS RNA viral polymerases is the presence of a unique enzymatic domain, called GDP polyribonucleotidyltransferase (PRNTase), which catalyzes pre-mRNA 5′-capping, one of the essential mRNA modifications. Our recent study revealed that the VSV PRNTase domain directs transcription initiation at the 3′-end of the genome as well as pre-mRNA capping with the dual functional priming-capping loop during stop-start transcription. Here, we further show that a helix structure flanked by the priming-capping loop regulates not only transcription elongation at an early phase of leader RNA synthesis but also polyadenylation-coupled transcription termination at gene junctions. These findings indicate that the PRNTase domain acts as a key regulatory domain for stop-start transcription as well as a catalytic domain for pre-mRNA capping.
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影响因子：
5.4
作者：
Barr, JN;Whelan, SPJ;Wertz, GW
通讯作者：
Wertz, GW
影响因子：
14.9
作者：
Neubauer J;Ogino M;Green TJ;Ogino T
通讯作者：
Ogino T
DOI：
10.1073/pnas.92.10.4477
发表时间：
1995-05-09
影响因子：
11.1
作者：
LAWSON, ND;STILLMAN, EA;ROSE, JK
通讯作者：
ROSE, JK
影响因子：
6.7
作者：
Braun, Molly R.;Deflube, Laure R.;Fearns, Rachel
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Fearns, Rachel
DOI：
10.1073/pnas.73.5.1504
发表时间：
1976-01-01
影响因子：
11.1
作者：
ABRAHAM, G;BANERJEE, AK
通讯作者：
BANERJEE, AK