Co-transcriptional Recruitment and Activity of the Spliceosomal snRNPs

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。 每个真核细胞的核心是细胞核，这是一个复杂的结构，高度组织成许多不同的功能域，以执行两个基本角色：基因组的维护和遗传信息的分布。 在亚核结构域中，基因被转录成前信使RNA（前mRNA）分子的位点。 当它们被合成时，前mRNA经历一系列复杂的加工事件以产生随后用于蛋白质合成的信使RNA。 其中一个剪接事件需要五个主要的小核糖核蛋白颗粒（snRNP）。 尽管snRNP在剪接中起着关键的催化作用，但对调节其募集为前mRNA的细胞机制仍知之甚少。 该计划旨在将snRNP的分析转移到两栖动物卵母细胞，这是一种独特的细胞系统，在透射光显微镜下可以直接观察单个转录位点和其他亚核结构域。 使用该体内系统的主要目的是确定snRNP与前mRNA相互作用及其共转录剪接活性所需的特征元件。 在青蛙卵母细胞中，活跃的转录位点对应于灯刷染色体（LBC）的侧环。这项研究是基于两个新开发的测定，这使得人们能够跟踪snRNP与LBC侧环的关联，并确定前体mRNA剪接是否分别发生在这些环上。 从长远来看，这些研究将有助于对控制活性转录单位的功能组织的细胞机制以及各种亚核结构域如何相互作用以调节前mRNA加工的一般理解。更广泛的影响两栖动物卵母细胞的巨大尺寸和易于操作使其特别适合于积极的外展计划。 事实上，向各级学生（本科生和高中生）展示在荧光显微镜下活跃转录的染色体和基因总是能成功地吸引他们的兴趣，同时向他们介绍实验科学的原理。 研究人员将利用这项研究继续促进与邻近高中和社区大学教师的关系，促进制定强有力的课程，并为包括妇女和少数民族在内的学生提供研究实习机会。 其目标是为教师提供科学支持，并让学生在学术环境中进行研究，同时促进他们从小型高中和社区学院过渡到大型本科校园。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Research project. At the heart of each eukaryotic cell is the nucleus, a complex structure highly organized into many distinct functional domains to perform two fundamental roles: the maintenance of the genome and the distribution of the genetic information. Among the sub-nuclear domains are the sites where genes are transcribed into pre-messenger RNA (pre-mRNA) molecules. As they are being synthesized, pre-mRNAs undergo a series of complex processing events to produce the messenger RNAs that are subsequently used for the synthesis of proteins. One of these events, splicing, requires the five major small ribonucleoprotein particles (snRNPs). Despite the critical catalytic role of snRNPs in splicing, little is still known about the cellular mechanisms that regulate their recruitment to pre-mRNAs. This program aims at moving the analysis of snRNPs to the amphibian oocyte, a unique cell system where direct visualization of individual transcription sites and other sub-nuclear domains is possible in the transmitted light microscope. The main objective, using this in vivo system, is to determine the characteristic elements of snRNPs required for their interactions with pre-mRNAs and their co-transcriptional splicing activity. In frog oocytes, the sites of active transcription correspond to the lateral loops of the lampbrush chromosomes (LBCs). The research is based on two newly developed assays, which permits one to follow the association of snRNPs with the lateral loops of LBCs and to determine whether pre-mRNA splicing occurs on these loops, respectively. In the long term, these studies will contribute to the general understanding of the cellular mechanisms that govern the functional organization of an active transcription unit, and how various subnuclear domains interact to regulate pre-mRNA processing.Broader Impacts.The giant size of an amphibian oocyte and its ease of manipulation make it particularly amenable to an active outreach program. Indeed, showing chromosomes and genes being actively transcribed in a fluorescence microscope to students of all levels (undergraduate and highschool) always succeeds at capturing their interest, while introducing them to the principles of experimental science. The investigator will use the research to continue fostering relations with teachers at neighboring high schools and community colleges, contributing to the development of strong curricula and providing their students, including women and minorities, with research internships. The goal is to provide teachers with scientific support and to expose students to research in an academic setting, while facilitating their transition from small high schools and community colleges to large undergraduate campuses.