Adeno-associated Virus RNA Splicing and Polyadenylation

描述（由申请人提供）：细小病毒RNA的内部聚腺苷酸化比以前认识到的更普遍，事实上，可以认为是规则而不是例外，作为控制细小病毒基因表达的手段。它可以发生在功能性内含子的内部或外部，但在所有情况下，内部多聚腺苷酸化阻止延伸到病毒衣壳蛋白编码基因中。因此，它是细小病毒基因表达的一个关键方面，了解其机制和影响至关重要。尽管所有AAV 5 RNA具有相同的单个内含子，但剪接相对于（pA）p处的内部多聚腺苷酸化的相对速率变化并且取决于5 '外显子的大小，即，RNA起始位点和内含子供体之间的距离。我们对AAV 5内部多聚腺苷酸化的初步表征使我们提出了一种模型，其中AAV 5 RNA的距离依赖性加工由U1 snRNP与非共有内含子供体的结合强度控制，其结合方式由5 '-外显子定义控制，通过与结合到RNA帽位点的核帽结合复合物（CBC）相互作用。该模型也可以解释AAV 2 RNA的距离依赖性剪接。在本申请的前两个具体目的中，我们提出严格测试我们的模型的两个主要步骤，即：i）AAV 5和AAV 2 5 ′-外显子的大小如何支配U1 snRNP与内含子供体的结合;和ii）U1 snRNP如何抑制AAV 5（pA）p处的多聚腺苷酸化。在第三个具体目标中，我们将确定允许AAV 5，而不是AAV 2，内部聚腺苷酸化，并确定这些差异如何影响它们的生命周期。在第四个具体目标中，我们将开始开发一种新的模型。我们将表征由红病毒B19（除AAV 5和非灵长类依赖病毒之外的唯一细小病毒）产生的RNA的内部聚腺苷酸化，以利用功能性内含子内的内部聚腺苷酸化。从拟议的研究中获得的知识将提供重要的洞察细小病毒基因表达的一个关键方面，并进入一般的外显子定义的机制。而且，正如过去一样，细小病毒分子遗传学的详细研究将继续为研究基因表达的基本分子机制提供有吸引力的，易处理的模型系统。公共卫生相关性：细小病毒是一种小的（20 nm）无包膜二十面体病毒，可感染许多脊椎动物宿主并引起疾病。它们也是用于基因治疗应用的非常有吸引力的载体。它们在所有已知的动物病毒中是独特的，因为它们含有单链线性DNA基因组。它们具有紧凑的遗传组织，其特征在于利用广泛的选择性剪接、选择性聚腺苷酸化和选择性翻译起始的重叠转录单位。我们已经开始在分子水平上表征细小病毒RNA的交替多聚腺苷酸化机制。我们建议在本提案中扩大这一分析。从我们的研究中获得的知识将促进我们对这一重要病毒组中基因表达的重要机制的理解。

DESCRIPTION (provided by applicant): Internal polyadenylation of parvovirus RNA is more prevalent than previously appreciated, and in fact, can be considered the rule rather than the exception as a means to govern parvovirus gene expression. It can occur either within or outside of a functional intron, but in all cases internal polyadenylation precludes extension into the virus capsid-coding gene. Thus, it is a key facet of parvovirus gene expression, and understanding its mechanism and impact are critical. Although all AAV5 RNAs have the same single intron, the relative rates of splicing vs internal polyadenylation at (pA)p varies and depends on the size of the 5'-exon, i.e., the distance between the RNA initiation site and the intron donor. Our initial characterization of AAV5 internal polyadenylation has led us to propose a model in which the distance-dependent processing of AAV5 RNA is controlled by the strength of U1snRNP binding to the nonconsensus intron donor in a manner governed by 5'-exon definition, via interaction with the nuclear cap-binding complex (CBC) bound to the RNA cap site. This model can also explain the distance-dependent splicing of AAV2 RNA. In the first two specific aims of this application, we propose to critically test the two major steps of our model, namely: i) how the size of the AAV5 and AAV2 5'-exon governs U1snRNP binding to the intron donor; and ii) how U1snRNP inhibits polyadenylation at AAV5 (pA)p. In the third specific aim we will determine the genetic differences that allow AAV5, but not AAV2, to internally polyadenylation, and determine how such differences impact their life cycles. In the fourth specific aim we will begin to develop a new model. We will characterize internal polyadenylation of RNA generated by the Erythrovirus B19, the only parvovirus other than AAV5 and the non-primate dependoviruses, to utilize internal polyadenylation within a functional intron. Knowledge gained from the proposed studies will provide important insight both into a critical aspect of parvovirus gene expression, and into the mechanism of exon definition in general. And, as has been true in the past, detailed studies of parvovirus molecular genetics will continue to provide attractive, tractable model systems for studying basic molecular mechanisms of gene expression. PUBLIC HEALTH RELEVANCE: Parvoviruses are small (20nm) non-enveloped icosahedral viruses that infect and cause disease in many vertebrate hosts. They are also highly attractive vehicles for gene therapy applications. They are unique among all known animal viruses in that they contain single- stranded linear DNA genomes. They have a compact genetic organization featuring overlapping transcription units which utilize extensive alternative splicing, alternative polyadenylation, and alternative translation initiation. We have begun to characterize the mechanism of alternative polyadenylation of parvovirus RNA at the molecular level. We propose to expand this analysis in this proposal. The knowledge gained from our studies will advance our understanding of this important mechanism of gene expression in this important group of viruses.