An Investigation into the Novel Role of PARP1 in Co-transcriptional Splicing

PARP1(PolyADP核糖聚合酶)是一种在多细胞动物中修复DNA损伤的重要蛋白质。PARP1参与了许多监测DNA完整性的生物过程，如细胞分裂、分化和细胞死亡。每个真核细胞中的DNA都是以高度紧凑的DNA-蛋白质结构的形式存在的，称为染色质，每次需要进入特定的DNA区域时，它都会被拆开，并在这个过程之后立即重新包装，以确保过程的保真度。已知PARP1在基因调控的启动过程中介导了这些染色质重排。最近的几项研究表明，PARP1在RNA剪接的不同过程中还有另一个意想不到的作用，即导致指导蛋白质合成的信使RNA正确形成的机制。这项研究将提供更好的理解PARP1如何调节选择性剪接来影响基因调控的变化。除了通过建立的知识库和研究结果的出版/展示向其他研究人员广泛提供研究结果外，该项目还将通过吸引研究生、本科生和高中生，特别是来自科学代表性不足的群体的学生，参与新兴的染色质生物学和基因调控领域的实验室研究，从而影响下一代劳动力。参与的学生将接受生物信息学、大规模基因组分析和染色质生物学方面的培训，并提供科学职业指导。高中生将完成小规模的实验室项目，并编写他们的第一个用于数据分析的计算机代码。在项目过程中，将制作五分钟的关于基因调控的电影剪辑，并将其纳入现有的生物化学课程。强有力的证据支持染色质在选择性剪接中的调节作用，染色质是细胞分化和发育的组成部分，有助于细胞谱系和组织认同。众所周知，反式因子(增强剂或抑制剂)与预先编码的mRNA序列元件的结合对于剪接调控是必要的，但不是充分的。本实验室最近发现，参与转录起始调控的染色质结构蛋白PARP1不仅结合在活性基因的转录起始点上，而且还结合在内部外显子上，与转录状态无关，这表明PARP1对基因的调控方式不同。此外，最近的发现表明，PARP1的缺失或其PAR化活性会导致选择性剪接事件的特定变化，并且在体内，PARP1结合染色质、新生mRNA和剪接因子。本研究将检验PARP1以两种非互斥方式调节共转录剪接的假设：作为适配器分子将剪接因子招募到外显子和/或以影响聚合酶延长和动力学的方式调节染色质结构的变化。将利用大规模基因组学和基因特异性方法来详细绘制发生在细胞中的PARP-1-RNA-染色质的遗传和生化相互作用图。由于相同的蛋白质存在于整个动物界的所有生物体中，PARP1很可能在简单和复杂的生物体中类似地调节替代决定。因此，这个问题将在一个易于进行遗传和分子分析的简单有机体--果蝇系统中进行研究。在实验室培养学生，特别是少数民族学生的历史基础上，这个项目将涉及当地的高中、本科生和研究生。此外，这些学校的少数族裔学生将被寻找在实验室进行研究的机会。这些人将接受生物信息学、大规模基因组分析和染色质生物学方面的培训。该项目的成果将以在地方、国家和国际会议上介绍的形式向更广泛的科学界传播。最后，这些研究的结果将发表在科学期刊上，并在科学会议上公布。

PARP1 (poly-ADP-ribose polymerase) is a protein important for repairing damaged DNA in multicellular animals. PARP1 is involved in many biological processes that monitor DNA integrity, such as cell division, differentiation and cell death. The DNA in every eukaryotic cell is found in the form of a highly compact DNA-protein structure known as chromatin, that is unpacked every time when access to a particular DNA region is needed, and re-packaged immediately after this process, to ensure the fidelity of the process. PARP1 is known to mediate these chromatin rearrangements during the initiation of gene regulation. Several recent studies show that PARP1 also has another, unexpected role in the different process of RNA splicing, i.e., the mechanism that results in the correct formation of the messenger RNAs that direct protein synthesis. This research will provide better understanding of how PARP1 modulates alternative splicing to effect changes in gene regulation. In addition to making the findings widely available to other researchers via established repositories and publication/ presentation of study outcomes, the project will impact the next-generation workforce by engaging graduate, undergraduate, and high school students, particularly students from groups underrepresented in the sciences, in laboratory research in the emerging field of chromatin biology and gene regulation. Participating students will be trained in bioinformatics, large-scale genome analyses, and chromatin biology and provided guidance on science careers. High school students will complete small-scale laboratory projects and write their first computer code for data analyses. During the course of the project, five-minute movie clips on gene regulation will be created and incorporated into an existing biochemistry course. Strong evidence supports the regulatory role of chromatin in alternative splicing, an integral part of cell differentiation and development, contributing to cell lineage and tissue identity. It is known that the binding of trans-factors (enhancers or silencers) to pre-mRNA-encoded sequence elements is necessary but not sufficient for splicing regulation. The recent discovery in this laboratory that PARP1, a chromatin architectural protein involved in the regulation of transcription initiation, not only bound at transcription start sites of active genes but also bound at internal exons, irrespective of the transcriptional state, suggests different modes of gene regulation by PARP1. Additionally, recent findings showed that depletion of PARP1 or its PARylation activity resulted in specific changes in alternative splicing events, and that in vivo PARP1 bound chromatin, nascent mRNA, and splicing factors. This research will test the hypothesis that PARP1 regulates co-transcriptional splicing in two non-mutually exclusive ways: acting as an adapter molecule to recruit splicing factors to exons and/or to modulate changes to chromatin structure in ways that affect polymerase elongation and kinetics. Large-scale genomics and gene-specific approaches will be utilized to map in fine detail the genetic and biochemical interactions of PARP-1-RNA-chromatin that occur in a cell. Since the same proteins are present in all organisms throughout the animal kingdom, PARP1 likely regulates alternative decisions similarly in simple and complex organisms. Therefore, this problem will be studied in a simple organism that is readily amenable to genetic and molecular analysis, the Drosophila system. Building on the laboratory's history of student training especially minority students, this project will involve local high schools, undergraduates, and graduate students. Additionally, minority students from these schools will be sought for research opportunities in the laboratory. These individuals will be trained in bioinformatics, large-scale genome analyses and chromatin biology. Results from this project will be disseminated to the broader scientific community in the form of presentations at local, national and international meetings. Finally, the results of these studies will be published in scientific journals and presented at scientific meetings.