The Role of Protein Arginine Methylation in the Co-transcriptional Recruitment of pre-mRNA Splicing Factors

智力上的功绩。在真核生物中，适当的基因表达对许多细胞过程和反应的控制至关重要。基因表达的最初步骤需要通过一个称为转录的过程将DNA复制到称为RNA的中间分子中。在转录过程中，未完成的RNA必须同时被许多特定的蛋白质处理，包括剪接因子(称为剪接体的大分子蛋白质复合体的成员)，以产生最终的成品。当RNA分子被制造和加工时，它们被包装成一种称为信使核糖核蛋白(MRNP)的复合体，一旦完成，整个mRNP就被从细胞核中输出出来。为了实现和协调这些生物过程的耦合，RNA结合蛋白必须在转录过程中以非常精确的顺序被招募到RNA中。作为一种扩展或调节蛋白质功能的方式，许多RNA结合蛋白是由特定的酶进行化学修饰的。精氨酸甲基化是一种在许多RNA结合蛋白上发现的化学修饰，这种修饰是由一系列被称为蛋白质精氨酸甲基转移酶(PRMT)的酶催化的。这个酶家族已经在从萌芽酵母到人类的各种物种中被发现，这个家族中最保守的成员是PRMT1。在萌芽酵母中，已证实酵母PRMT1(称为Hmt1)在转录过程中促进剪接因子的招募，这对mRNP的形成至关重要。本项目的目标是研究蛋白质精氨酸甲基化在简单真核模式生物酿酒酵母中调节剪接因子共转录招募的机制。将综合运用分子生物学、生物化学、蛋白质组学和基因组学方法。由于酵母Hmt1及其同源物在高等真核生物中的高度保守性，从这个项目中获得的信息将提供可能适用于这些组织的见解。该项目将为布法罗大学提供培训研究生和博士后研究员的机会，布法罗大学是一个非常多样化的校园。研究生将接受分子生物学、生物化学和基因组学领域的培训。本科生也将被招募参加这个项目的各个方面，这些新成员将包括纽约大学科学与技术进入计划(CSTEP)的成员，这是一个为经济上处于不利地位或历史上代表不足的学生而设的丰富计划。由于该项目涉及将分子生物学与基因组学和计算分析相结合，学生将从跨学科培训中受益。旅行资金将用于允许学生在国家科学会议上发表演讲和张贴海报。这项工作的发现将被纳入高级分子生物学实验室课程，提供给布法罗大学更高年级的本科生。这个项目产生的微阵列数据将向公众开放。

Intellectual Merit. In eukaryotes, proper gene expression is vital for the control of many cellular processes and responses. The initial step of gene expression requires the copying of the DNA into an intermediate molecule called the RNA via a process called transcription. During transcription, the unfinished RNA must simultaneously be processed by a multitude of specific proteins, including the splicing factors (which are members of a macromolecular protein complex called the spliceosome), in order to generate a final, finished product. As the RNA molecules are made and processed, they are packaged into a complex called messenger ribonucleoprotein (mRNP) particle and once finished, the entire mRNP is then exported out of the cell nucleus. To achieve and coordinate the coupling of these biological processes, the RNA-binding proteins must be recruited to the RNA in a very precise order during transcription. As a way to expand or to regulate the function of a protein, many RNA-binding proteins are modified chemically by specific enzymes. Arginine methylation is one such type of chemical modification that is found on many RNA binding proteins, and this modification is catalyzed by a family of enzymes known as protein arginine methyltransferases (PRMT). This enzyme family has been identified in species ranging from the budding yeast to humans, with the most conserved member of this family being PRMT1. In the budding yeast Saccharomyces cerevisiae, it was established that yeast PRMT1 (termed Hmt1) plays a functional role in promoting the recruitment of splicing factors during transcription, which is vital to mRNP formation. The goals of this project are to examine the mechanisms by which protein arginine methylation modulates the co-transcriptional recruitment of splicing factors in the simple eukaryotic model organism Saccharomyces cerevisiae. A combination of molecular biology, biochemistry, proteomic, and genomic methodologies will be used. Because of the high conservation between yeast Hmt1 and its homologs in higher eukaryotes, the information obtained from this project will provide insights that will likely be applicable to those organisms.Broader Impacts. This project will provide opportunities to train graduate students and post-doctoral fellows at University at Buffalo, which is a very diverse campus. The graduate students will receive training in the areas of molecular biology, biochemistry, and genomics. Undergraduate students will also be recruited to take part in various aspects of this project and these recruits will include members of the New York Collegiate Science & Technology Entry (CSTEP) program, an enrichment program for students who are economically disadvantaged or historically underrepresented. Since the project involves the use of molecular biology with genomics and computational analysis, the students will benefit from crossdisciplinary training. Travel funds will be used to allow students to present talks and posters at national scientific meetings. Findings from this work will be incorporated into an Advanced Molecular Biology laboratory course offered to the more senior undergraduates at University at Buffalo. The microarray data generated from this project will be made available to the public.