Mechanism of U1 snRNPs suppression of premature cleavage & polyadenylation

描述(由申请人提供)：这项提案的首要目标是了解远程转录的机制，这是我的实验室最近发现的一种新的和主要的基因表达过程。在真核生物中合成信使RNA(信使RNA)所必需的大多数蛋白质编码基因的转录中，端粒转录对于全长RNA聚合酶II(PolII)是必不可少的。它依赖于U1 SnRNP(U1)，这是一个丰富的非编码11亚基核糖核蛋白颗粒，可以保护新生的PolII转录本不会通过内含子的切割和多聚腺苷酸化(CPA)而提前终止，这是高度破坏性的。U1 SnRNP在5‘剪接位点(Ss)识别中的作用是很好的特征，它是内含子剪接的关键和第一步。然而，我们发现U1具有额外的非剪接功能，可以抑制随机存在于大内含子中的隐蔽多聚腺苷酸化信号(PASS)中的早熟CPA(PCPA)。我们将U1‘S PCPA抑制也称为远程转录，因为这是允许转录走得更远所必需的。相比之下，来自不同polII启动子的新生上游反义转录本由于与PAS和U1的结合比率相反，相对不受保护，并迅速降解，表明远程转录在塑造转录组中起着普遍的作用。此外，远端转录活性也决定了mRNA的长度，远端转录活性可以通过U1水平的微小变化进行精细调节。例如，U1的轻微减少会导致广泛的缩短，因为在3‘非翻译区(3’UTR)使用了更近端的PASS，从而消除了翻译抑制microRNA结合位点等mRNA调节元件。基于我们以前的研究，我们提出了一个模型来解释U1如何抑制内含子中的PASS，包括它与内含子结合的假设，而不仅仅是与5‘s结合。然而，检验这一假说和理解远程脚本机制所需的基本信息是缺乏的。为了解决这个问题，我的实验室建立了精确操作PCPA及其被U1抑制的工具，并对其进行了详细的检测。我们建议追求三个特定的目标：1)在活细胞的新生转录本上生成U1 SnRNP结合位点的全基因组图谱，该图谱将相对于我们最近完成的PCPA位点全基因组图谱进行解释。2)确定U1SnRNP蛋白和必需的U1SnRNA结构域(S)在远转录中的作用。3)利用本实验室首创的发现RNP相互作用组的综合方法，识别CPA复合体和/或其他抑制U1‘S PCPA的靶点。综上所述，这些目标将为U1 SNRNP远程转录的机制提供重要的见解，并促进对这一新的基因调控维度在生物学和医学中的理解和潜在应用。

DESCRIPTION (provided by applicant): This proposal's overarching goal is to understand the mechanism of telescripting, a new and major gene expression process recently discovered in my laboratory. Telescripting is essential for full length RNA polymerase II (pol II) transcription from the majority of protein-coding genes in eukaryotes necessary for messenger RNA (mRNA) synthesis. It relies on U1 snRNP (U1), an abundant non-coding 11-subunit ribonucleoprotein particle, to protect nascent pol II transcripts from early termination by cleavage and polyadenylation (CPA) in introns, which is highly destructive. U1 snRNP is well characterized for its role in 5' splices site (ss) recognition, a key and the first step in splicing of introns. Howeer, we found that U1 has an additional non-splicing function as a suppressor of premature CPA (PCPA) from cryptic polyadenylation signals (PASs) that are stochastically present in large introns. We refer to U1's PCPA suppression also as telescripting, as it is necessary for allowing transcription to go farther. In contrast, nascent upstream antisense transcripts from divergent polII promoters are relatively unprotected due to an inverse PAS to U1 binding ratio and are rapidly degraded, indicating telescripting's general role in shaping the transcriptome. Furthermore, telescripting activity, which can be finely modulated by slight changes in U1 level also determines mRNA length. For example, slight U1 decrease causes widespread shortening due to usage of more proximal PASs in the 3' untranslated region (3'UTR), thereby removing mRNA-regulating elements such as translation repressing microRNA binding sites. Based on our previous studies we proposed a model to explain how U1 suppresses PASs in introns, including the hypothesis that it binds in introns and not only to 5'ss. However, basic information needed to test this hypothesis and for understanding telescripting mechanism is lacking. To address this, my laboratory established tools to precisely manipulate PCPA and its suppression by U1, and assays to probe it in detail. We propose to pursue three specific aims: 1) To generate genome wide map of U1 snRNP binding sites on nascent transcripts in live cells, which will be interpreted relative to genome wide map of PCPA sites that we recently completed. 2) To determine the role of U1 snRNP proteins and essential U1 snRNA domain(s) for telescripting. 3) To identify CPA complex and/or other targets of U1's PCPA suppression using a comprehensive RNP interactome discovery approach pioneered in my laboratory. Together, these aims will provide important insights into mechanism of U1 snRNP telescripting, and advance understanding and potential applications of this new dimension in gene regulation to biology and medicine.