Uncovering the mechanisms of splice site choice

揭示剪接位点选择的机制

• 批准号: 1613867
• 负责人: Alan Zahler
• 金额: $ 56.5万
• 依托单位: University of California-Santa Cruz
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1613867&HistoricalAwards=false
• 关键词: Uncovering; mechanisms; splice; site; choice

This project is aimed at understanding how the genetic information stored in DNA is accurately processed to code for protein assembly. In order for information in DNA to be used in the cell, the DNA is first copied into an intermediate sister molecule called RNA which is then decoded into protein sequence. However, in order to be useful, internal parts of the RNA must first be removed and the surrounding sequences precisely pasted together by a process called RNA splicing. Splicing is a carried out by one of the largest and most complicated machines in the cell, the spliceosome, which consists of over 100 proteins and 5 RNA molecules that assemble onto the RNA at the region to be spliced out. Because of the complexity of assembling such a large machine onto the RNA, there is still much to learn about the mechanisms by which the spliceosome accurately chooses the sites in the RNA that will be spliced together. Genetic experiments to search for mutations that affect splice site choice are an important and cutting-edge component of this research, and these experiments will be carried out by minority undergraduate participants in an established summer undergraduate training program at the University of California-Santa Cruz (UCSC). This training will be important in preparing these students for positions in UCSC faculty research labs, and for future admission into PhD programs.This project employs the microscopic worm Caenorhabditis elegans as a model to explore pre-mRNA splicing through integrated genetic, genomic and biochemical approaches. Unique approaches have been developed in the preliminary studies that uncovered new regulatory phenomena for splice site selection at both ends of the region of RNA to be removed, called the 5' and 3' splice sites. Genetic screens have been used to uncover new roles for factors important for the regulation of 5' splice site choice. Fundamental differences have been uncovered in alternative adjacent 3' splice site selection between somatic and germ cells. The research will expand on these observations by addressing the following two questions: how do protein factors regulate the choice of 5' splice sites, and what are the mechanistic differences in 3' splice site selection between soma and germ cells? The results are expected to provide novel insights into how the splicing machinery accurately chooses the 5' and 3' splice sites.

该项目的目的是了解如何准确处理中存储在DNA中的遗传信息以编码蛋白质组装。为了在细胞中使用DNA中的信息，首先将DNA复制成称为RNA的中间姐妹分子，然后将其解码为蛋白质序列。但是，为了有用，必须首先去除RNA的内部部分，并通过称为RNA剪接的过程将周围序列精确地粘合在一起。剪接是由细胞中最大，最复杂的机器之一进行的剪接，该机器由100多种蛋白质和5个RNA分子组成，这些蛋白质和5个RNA分子在该区域的RNA聚集在该区域，以示插入。由于将如此大的机器组装到RNA上的复杂性，仍然需要了解剪接体准确选择RNA中将剪接在一起的位点的机制。寻找影响剪接场所选择的突变的基因实验是这项研究的重要组成部分，这些实验将由少数群体本科参与者在加利福尼亚大学 - 圣克鲁斯大学（UCSC）的一项既定的夏季本科培训计划中进行。这项培训对于使这些学生在UCSC教职研究所的职位以及将来进入博士学位课程的职位方面将很重要。该项目采用微观蠕虫Caenorhabditis elegans作为模型来探索通过综合基因组，基因组和生物化学方法来探索MRNA的剪接。在初步研究中已经开发出了独特的方法，该研究发现了在要去除的RNA区域的两端的新调节现象，用于剪接位点选择，称为5'和3'剪接位点。遗传筛选已被用来发现新的作用，以调节5'剪接位点选择的重要因素。在体细胞和生殖细胞之间的替代3'剪接位点选择中，已经发现了基本差异。这项研究将通过解决以下两个问题来扩展这些观察结果：蛋白质因子如何调节5'剪接位点的选择，以及SOMA和生殖细胞之间3'剪接位点选择的机械差异是什么？ 预计结果将提供有关剪接机械如何准确选择5'和3'剪接位点的新见解。