Mechanism of U1 snRNPs suppression of premature cleavage & polyadenylation

描述（由申请人提供）：本提案的首要目标是了解转录机制，这是我实验室最近发现的一种新的主要基因表达过程。套刻对于真核生物中信使RNA（mRNA）合成所需的大多数蛋白质编码基因的全长RNA聚合酶II（pol II）转录至关重要。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，以保护新生的pol II转录物免受内含子中的切割和多聚腺苷酸化（CPA）的早期终止，这是高度破坏性的。U1 snRNP在5'剪接位点（ss）识别中的作用是内含子剪接的关键和第一步。然而，我们发现U1还有一个额外的非剪接功能，作为来自随机存在于大内含子中的隐蔽多聚腺苷酸化信号（PAS）的过早CPA（PCPA）的抑制因子。我们也将U1的PCPA抑制称为转录抑制，因为它是允许转录进一步进行的必要条件。相比之下，新生上游反义转录从不同的polII启动子是相对不受保护的，由于反向PAS U1的结合比，并迅速降解，表明转录的一般作用，在形成的转录组。此外，转录活性也决定了mRNA的长度，它可以通过U1水平的轻微变化进行精细调节。例如，由于在3'非翻译区（3' UTR）中使用更近端的PAS，轻微的U1减少导致广泛的缩短，从而去除mRNA调节元件，例如翻译抑制microRNA结合位点。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PAS，包括它结合在内含子中而不仅仅是5 'ss的假设。然而，测试这一假设所需的基本信息和理解的转录机制是缺乏的。为了解决这个问题，我的实验室建立了精确操纵PCPA及其被U1抑制的工具，并进行了详细的检测。我们提出了三个具体的目标：1）在活细胞中新生转录本上生成U1 snRNP结合位点的全基因组图谱，这将相对于我们最近完成的PCPA位点的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)为了确定CPA复合物和/或U1的PCPA抑制的其他目标，使用在我的实验室开创的全面RNP相互作用组发现方法。总之，这些目标将提供重要的洞察机制的U1 snRNP转录，并推进理解和潜在的应用，在生物学和医学基因调控的这一新的层面。

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.