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加工事件共转录地发生，由与延伸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。预期基因表达的病毒调节剂将影响这一过程，然而，迄今为止，这种病毒调节剂的例子很少。我们最近已经证明，在腺病毒5型（Ad 5）的存在下，腺相关病毒2型（AAV 2），细小病毒科的一个成员，作为一个共同的转录的RNA加工的反式调节蛋白的大Rep蛋白。我们建议进一步表征Rep在Specific Aim I中的这种新活性，通过：1.确定AAV 2转录单位中的顺式作用信号，其是响应Rep和Ad 5的RNA加工效应所必需的并且是足够的; 2.确定支持Rep效应所必需的反式作用辅助功能，并且通过确定介导其RNA加工效应的Rep蛋白的结构域; 3.确定Rep在AAV 2 RNA加工中的共转录作用机制。具体来说，我们将测试我们的模型，即Rep改变与延伸转录复合物的CTD相关的RNA加工因子的组成。我们对其他AAV血清型的转录谱的调查已经揭示，从AAV 5产生的RNA有效地利用了病毒内含子内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。这将在具体目标2中通过以下方式进一步表征：1.表征控制AAV 5 RNA的选择性多聚腺苷酸化的顺式和反式作用决定簇; 2.确定为什么从上游P7启动子产生的RNA利用（pA）p，而从附近P41读取的RNA利用（pA）p;和3.检查内部多聚腺苷酸化对AAV 5生命周期的影响。基因表达的这些方面是AAV生物学的至关重要的特征。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，因此它们在易于处理的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.