U12-Dependent Spliceosomal snRNAs

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。真核生物细胞核中通过剪接从前体rna （pre）到信使rna (m)去除非编码片段内含子是基因表达的重要步骤。两阶段剪接反应是由一个被称为剪接体的蛋白质和RNA的大复合体进行的。许多后生动物含有两种类型的内含子，它们在边界处具有不同的序列（GU-AG或AU-AC），并被含有不同小核rna （snrna）的剪接体去除。根据识别分支点的剪接体snRNA，主要类别的内含子被指定为依赖于u2，次要类别的内含子被指定为依赖于u12。尽管存在十亿年的差异，但不仅是基本的化学过程，而且剪接所需的许多RNA-RNA相互作用在主要剪接体和次要剪接体之间都是保守的。尽管在rna的排列和剪接位点识别事件中存在显著的相似之处，但在两种剪接系统中只有一个snRNA是共同的。最近，研究表明，U4 snRNA可以与u6攻击snRNA进行碱基对，从而在体内剪接u12依赖的内含子，这就提出了一个有趣的问题，即为什么在主要剪接体和次要剪接体中存在两组不同的snRNA。在这个项目中，这个问题将通过探索精细调整的RNA-RNA相互作用来解决，这种相互作用是u12依赖性剪接体所特有的。由于许多分子间RNA-RNA相互作用似乎是类似的，并且在两个剪接体之间包含可互换的功能元件，因此本研究将解决主要剪接体和次要剪接体snrna之间的进化关系。此外，将定义确保选择各自的snrna集以形成适当的核糖核蛋白复合物的机制。该研究项目将继续开发基因抑制系统，以更好地了解含有u12剪接体的结构和功能之间的关系，从而揭示有关植物和动物王国中依赖u12的内含子和相应剪接体的起源和保留的进化问题。更广泛的影响：本研究的更广泛的影响包括加强对两个剪接体共存的起源和意义的理解。从拟议的研究中获得的结果将通过由首席研究员教授的讲座和实验课程传达给本科生和研究生。这项研究将通过在一个密集的、积极的学习环境中向从事该项目的本科生和研究生介绍RNA分子生物学方法，从而影响他们。在外联活动方面，该项目将主持和促进涉及高中学生和弱势少数族裔以及社区大学非少数族裔学生的研究。这些学生将有机会在实验室进行独立研究，从而提高他们长期从事研究工作的前景。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)Removal of non-coding segments known as introns from precursor (pre) to messenger (m) RNAs in eukaryotic nuclei via splicing is an essential step in gene expression. The two-ste splicing reaction is carried out by a large complex of protein and RNA known as the spliceosome. Many metazoans contain two types of introns that have distinct sequences at their boundaries (GU-AG or AU-AC) and are removed by spliceosomes that contain different sets of small nuclear RNAs (snRNAs). The major class of introns is designated U2-dependent and the minor class U12-dependent according to the spliceosomal snRNA that recognizes the branchpoint. Despite a billion years of divergence, not only the fundamental chemical processes, but many of the RNA-RNA interactions required for splicing are conserved between the major and minor spliceosomes. Although significant parallels exist in the arrangement of the RNAs and in the splice site recognition events, only one snRNA is common between the two splicing systems. Recently, it was demonstrated that U4 snRNA can base pair with U6atac snRNA to splice a U12-dependent intron in vivo, raising the interesting question as to why two distinct sets of snRNAs are present in the major and minor spliceosomes. In this project, this question will be addressed by exploring finely tuned RNA-RNA interactions that are unique to the U12-dependent spliceosome. Since a number of intermolecular RNA-RNA interactions appear to be analogous and contain interchangeable functional elements between the two spliceosomes, the study will address evolutionary relationships between the major and the minor spliceosomal snRNAs. Furthermore, the mechanisms that ensure the selection of respective sets of snRNAs to form the appropriate ribonucleoprotein complexes will be defined. The research project will continue to exploit genetic suppression systems to achieve a greater understanding of the relationship between structure and function within the U12-containing spliceosome, thereby shedding light on evolutionary questions regarding the origin and retention of U12-dependent introns and the correspondings spliceosomes in the plant and animal kingdoms.Broader impacts: The broader impacts of this research include an enhanced understanding of the origin and significance of the co-existence of two spliceosomes. Results obtained from the proposed research will be communicated to undergraduate and graduate students through lecture and laboratory courses taught by the principal investigator. The research will influence undergraduate and graduate students working on the project by introducing them to RNA molecular biology methods in an intensive, active learning environment. In terms of outreach activities, this project will host and promote research involving high school students and underrepresented minority as well as non-minority students from a community college. These students will be given the opportunity to undertake independent research studies in the laboratory, thereby enhancing their prospects for long-term careers in research.