Mechanism of U1 snRNPs suppression of premature cleavage & polyadenylation

描述（由申请人提供）：本提案的总体目标是了解电转录的机制，这是我实验室最近发现的一种新的主要基因表达过程。在真核生物中，信使RNA （mRNA）合成所必需的大多数蛋白质编码基因转录全长RNA聚合酶II （pol II）是必不可少的。它依赖于U1 snRNP (U1)，一个丰富的非编码11亚基核糖核蛋白颗粒，保护新生的pol II转录本免受内含子裂解和聚腺苷化（CPA）的早期终止，这是高度破坏性的。U1 snRNP在5‘剪接位点（5’ splices site, ss）识别中发挥着重要作用，这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，作为来自大内含子随机存在的隐聚腺苷化信号（PASs）的过早CPA （PCPA）的抑制因子。我们将U1的PCPA抑制也称为电转录，因为它是允许转录进一步进行的必要条件。相比之下，来自不同的polII启动子的新生上游反义转录物由于PAS与U1的反向结合比而相对不受保护，并且会迅速降解，这表明转录在塑造转录组中的一般作用。此外，可以通过U1水平的微小变化精细调节的转录活性也决定了mRNA的长度。例如，U1的轻微降低会导致3‘非翻译区（3’ utr）中更多近端PASs的使用，从而导致广泛的缩短，从而去除mrna调节元件，如翻译抑制microRNA结合位点。基于我们之前的研究，我们提出了一个模型来解释U1如何抑制内含子中的PASs，包括它结合在内含子而不仅仅是5's上的假设。然而，检验这一假设和理解电抄写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纵PCPA及其U1抑制的工具，并进行了详细的检测。我们提出了三个具体目标：1)生成活细胞新生转录本上U1 snRNP结合位点的全基因组图谱，并将其与我们最近完成的PCPA位点全基因组图谱进行解释。2)确定U1 snRNP蛋白和必需的U1 snRNA结构域在转录中的作用。3)使用我实验室首创的综合RNP相互作用组发现方法鉴定U1的CPA复合物和/或其他PCPA抑制靶点。总之，这些目标将为U1 snRNP转录机制提供重要的见解，并促进对这一基因调控新维度的理解和潜在应用于生物学和医学。

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.