Mechanism of U12-dependent spliceosomal splicing

描述（由申请人提供）：通过剪接从信使RNA前体中去除内含子是高等真核生物大多数基因表达的必经步骤。先前的工作已经定义了在大多数后生动物门的基因组中发现的一类罕见的内含子（依赖于u12的内含子）。这类内含子存在于大约1%的人类基因中，包括许多具有基本和/或疾病相关功能的基因。依赖于u12的内含子和更丰富的依赖于u2的内含子都是通过类似的机制在被称为剪接体的大而复杂的核糖核蛋白结构中剪接。这两种剪接体的snRNA组成不同，但剪接体核心的RNA-RNA相互作用有显著的相似之处。这些高度保守的序列和相互作用可能构成剪接体的功能元件。此外，许多参与剪接的蛋白质似乎在这两个系统中是共享的，但不是全部。为了了解这些内含子在人类基因表达和剪接的生物化学中的作用，需要详细了解这种第二剪接系统的剪接信号和机制。本应用程序旨在研究这类次要内含子的剪接机制。首先，RNA干扰技术将用于筛选果蝇和哺乳动物细胞中次要类剪接所需的蛋白质因子。其次，提出了实验来研究剪接体形成的特异性。已经确定了控制剪接体RNA与u12依赖性内含子相互作用的RNA元件。这些元素的功能及其相互作用的因素将被研究。第三，将确定小类snrna的关键功能特征，包括探索这些snrna与自剪接II组内含子元件之间的关系。公共卫生相关性：基因的调控表达对人类生长、发育、正常和病理功能以及身体对内外环境变化的反应至关重要。基因调控的一个重要方面是通过RNA剪接从基因的初级转录本中去除内含子。该建议旨在了解如何正确选择剪接位点，以及正确的RNA片段如何在人类基因的最终mRNA产物中连接在一起。

DESCRIPTION (provided by applicant): The removal of introns from messenger RNA precursors by splicing is a mandatory step in the expression of most genes of higher eukaryotes. Previous work has defined a rare class of introns (U12-dependent introns) found in the genomes of most metazoan phyla. Members of this intron class are present in about 1 % of human genes including many with essential and/or disease-associated functions. Both the U12-dependent introns and the more abundant U2-dependent introns are spliced via a similar mechanism in large and complex ribonucleoprotein structures called spliceosomes. The two spliceosomes differ in their snRNA composition yet share significant similarities in the RNA-RNA interactions at the spliceosome core. These highly conserved sequences and interactions likely constitute the functional elements of the spliceosome. In addition, many but not all of the proteins involved in splicing appear to be shared between the two systems. A detailed understanding of the splicing signals and mechanism of this second splicing system is needed to understand the role of these introns in human gene expression and the biochemistry of splicing. This application proposes to examine the mechanism of splicing of this minor class of introns. First, RNA interference techniques will be used to screen for protein factors required for minor class splicing in Drosophila and mammalian cells. Second, experiments are proposed to investigate the specificity of spliceosome formation. RNA elements have been identified that control which spliceosomal RNAs interact with U12-dependent introns. The function of these elements and the factors they interact with will be investigated. Third, the critical functional features of minor class snRNAs will be identified including the exploration of relationships between these snRNAs and elements of self-splicing group II introns. Public health relevance: The regulated expression of genes is central to human growth, development, normal and pathological functioning and the response of the body to changes in the internal and external environment. An important point of gene regulation is the removal of introns from the primary transcripts of genes by RNA splicing. This proposal is designed to understand how the sites of splicing are correctly chosen and how the correct RNA fragments are linked together in the final mRNA products of human genes.