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加工事件发生共转录，由与延长RNA pol II复合物的羧基末端结构域（CTD）相关的RNA加工因子介导。可以预期，基因表达的病毒调节因子会影响这一过程，然而，迄今为止，这种病毒调节因子的例子很少被发现。我们最近证明，在5型腺病毒（Ad5）存在的情况下，细小病毒科成员腺相关病毒2型（AAV2）的大Rep蛋白作为RNA加工的共转录反式调节因子。我们建议在特异性Aim I中进一步表征Rep的这种新活性，通过：1.确定AAV2转录单元中的顺式作用信号，这些信号是对Rep和Ad5的RNA加工效应作出反应所必需的和足够的；2.确定支持Rep作用所需的反式辅助功能，并通过确定介导其RNA加工作用的Rep蛋白结构域；3.确定Rep共转录对AAV2 RNA加工的作用机制。具体来说，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血清型转录谱的调查显示，由AAV5产生的RNA有效地利用了病毒内含子内的聚腺苷化位点（pA） p。（pA） p的使用与启动子与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。这将在具体目标2中进一步说明：1。表征控制AAV5 RNA选择性聚腺苷化的顺式和反式决定因素；2.确定为什么上游P7启动子产生的rna利用（pA） p，而附近P41启动子产生的rna读取；和3。研究内部聚腺苷化对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加工功能可能在共转录框架中得到最好的理解，因此它们在可处理的AAV系统中的表征将有助于阐明这种基因表达的新范式。

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.