Adeno-associated Virus RNA Splicing and Polyadenylation

描述(由申请人提供)：越来越清楚的是，在体内，RNA加工事件是由与延长的RNA PolII复合体的羧基末端结构域(CTD)相关的RNA加工因子介导的共转录发生的。预计病毒对基因表达的调节会影响这一过程，然而，到目前为止，这种病毒调节的例子还很少。我们最近证实，在5型腺病毒(Ad5)存在的情况下，腺相关病毒2型(AAV2)的大Rep蛋白(AAV2)是细小病毒科(Parvoviridae)家族的成员，扮演着RNA加工的共转录反式调节因子的角色。我们建议在特定目的I中进一步表征Rep的这一新活性，方法是：1.确定AAV2转录单元中对Rep和Ad5的RNA加工效应做出反应所需且足够的顺式作用信号；2.确定支持Rep效应所需的反式作用辅助功能，并确定介导其RNA加工效应的Rep蛋白的结构域；以及3.确定Rep对AAV2 RNA加工的共转录效应的机制。具体地说，我们将测试我们的模型，即Rep改变与延长转录复合体的CTD相关的RNA加工因子的组成。我们对其他AAV血清型的转录图谱的调查表明，AAV5产生的RNA有效地利用了病毒内含子中的多聚腺苷酸化位点(PA)p。(Pa)p的使用与启动子和该位点之间的距离成正比，并且(Pa)p受到上游内含子供体的抑制。这将在特定目标2中进一步表征，方法是：1.表征控制AAV5 RNA选择性多聚腺化的顺式和反式作用决定因素；2.确定为什么从上游P7启动子产生的RNA利用(Pa)p，以及来自附近P41的RNA通读；以及3.检查内部多腺基化对AAV5生命周期的影响。基因表达的这些方面是AAV生物学的重要特征。此外，由于这两种RNA处理功能可能都在共转录框架中得到了最好的理解，它们在易处理的AAV系统中的特征将有助于阐明这种新的基因表达范式。

DESCRIPTION (provided by applicant): It has become increasingly clear that, in vivo, RNA processing events occur co-transcriptionally, mediated by RNA processing factors associated with the carboxy-terminal domain (CTD) of the elongating RNA pol II complex. It would be expected that viral regulators of gene expression would influence this process, however, few examples of such viral regulators have so far been identified. We have recently demonstrated that in the presence of adenovirus type 5 (Ad5), the large Rep protein of adeno-associated virus type 2 (AAV2), a member of the Parvoviridae Family, acts as a co-transcriptional trans-regulator of RNA processing. We propose to further characterize this novel activity of Rep in Specific Aim I, by: 1.determining the cis-acting signals in the AAV2 transcription unit that are required and sufficient to be responsive to the RNA processing effects of Rep and Ad5; 2.determining the trans-acting helper functions that are required to support Rep's effect, and by determining the domains of the Rep protein that mediate its RNA processing effect; and 3.determining the mechanism of Rep's co-transcriptional effect on processing of AAV2 RNA. Specifically, we will test our model that Rep alters the composition of RNA processing factors associated with the CTD of the elongating transcription complex. Our survey of the transcription profile of the other AAV serotypes has revealed that RNA produced from AAV5 efficiently utilizes a polyadenylation site, (pA) p, within the viral intron. The usage of (pA) p is directly proportional to the distance between the promoter and that site, and (pA) p is subject to inhibition by an upstream intron donor. This will be further characterized in Specific Aim 2, by: 1.characterizing the cis-and trans-acting determinants that govern the alternative polyadenylation of AAV5 RNA; 2.determining why RNAs generated from the upstream P7 promoter utilize (pA) p, and those from nearby P41 read through; and 3.examining the consequences of internal polyadenylation for the AAV5 life cycle. These aspects of gene expression are critically important features of AAV biology. Also, as both of these RNA processing functions are probably best understood in a co-transcriptional framework, their characterization in the tractable AAV system will help illuminate this new paradigm of gene expression.