Adeno-associated Virus RNA Splicing and Polyadenylation

说明书(申请人提供)：细小病毒RNA的内部多聚腺苷化比以前所认识的更普遍，事实上，可以被认为是规则而不是例外作为控制细小病毒基因表达的一种手段。它既可以发生在功能内含子内，也可以发生在功能内含子外，但在所有情况下，内部多聚腺苷基化都会阻止病毒衣壳编码基因的延伸。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。虽然所有的AAV5 RNA都有相同的单个内含子，但(PA)p处剪接和内部多聚腺苷基化的相对速率是不同的，并且取决于5‘-外显子的大小，即RNA起始点和内含子供体之间的距离。我们对AAV5内部多聚腺苷基化的初步表征导致我们提出了一个模型，在该模型中，AAV5 RNA的距离依赖于加工，该模型通过与RNA帽部位结合的核帽结合复合体(CBC)的作用，以5‘-外显子定义的方式控制U1SnRNP与非共识内含子供体的结合强度。这个模型也可以解释AAV2 RNA的距离依赖的剪接。在本申请的前两个特定目标中，我们建议对我们模型的两个主要步骤进行关键测试，即：i)AAV5和AAV2 5‘-外显子的大小如何调控U1SnRNP与内含子供体的结合；以及ii)U1SnRNP如何抑制AAV5(PA)p的多腺基化。在第三个特定目标中，我们将确定允许AAV5而不是AAV2内部多腺基化的遗传差异，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式。我们将表征由红血球病毒B19产生的RNA的内部多腺化作用，以利用功能内含子中的内部多腺化作用。从拟议的研究中获得的知识将为了解细小病毒基因表达的关键方面以及一般情况下外显子定义的机制提供重要的见解。而且，正如过去的情况一样，对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：细小病毒是小的(20 Nm)无包膜的二十面体病毒，在许多脊椎动物宿主中感染并导致疾病。它们也是基因治疗应用的极具吸引力的载体。它们在所有已知的动物病毒中是独一无二的，因为它们包含单链线性DNA基因组。它们具有紧凑的遗传组织，以重叠的转录单位为特征，利用广泛的选择性剪接、选择性多聚腺苷化和选择性翻译起始。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的机制。我们建议在本提案中扩展这一分析。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这一重要机制的理解。

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.