DNA transposons and alternative pre-mRNA splicing

 描述（由申请人提供）：在所有生物体的基因组中发现了移动的遗传元件或转座子。这些元素可以通过DNA或RNA中间体移动。约50%的人类基因组由转座因子组成，其中约2.7%对应于基于DNA的转座子。许多这些假定的转座子或转座酶相关基因是未知的。我们以前的研究主要集中在果蝇中的P元件家族的DNA转座子。P元件转座酶以四聚体的形式起作用，使用GTP作为转座的辅因子。转座酶的N-末端结构域对应于C2CH THAP DNA结合结构域，其是仅在动物基因组中发现的DNA结合结构域的普遍家族的成员。一个THAP基因，称为THAP 9，与果蝇P元件转座酶同源，存在于灵长类动物、非洲爪蟾、斑马鱼和玻璃海鞘中，但在啮齿动物中不存在。我们实验室最近的工作表明，人类和斑马鱼的THAP 9基因可以动员果蝇和斑马鱼的P元件转座子在人类和果蝇细胞。本研究旨在了解人THAP 9基因在人胚胎干细胞中的作用，以及果蝇P元件转座酶蛋白如何识别并与转座子末端、供体DNA、靶DNA和GTP/Mg 2+组装形成活性蛋白-DNA复合物。这些研究旨在获得机械的见解。 前体mRNA选择性剪接是后生动物基因表达调控的重要机制，是基因组序列转化为蛋白质组信息的通道。大多数真核基因是分裂的，并具有选择性剪接的潜力，大大增加了蛋白质组的多样性。许多人类和小鼠疾病基因突变影响剪接过程。剪接沉默子是一种主要类型的RNA控制元件，产生组织或细胞类型特异性的选择性剪接模式。我们以前的工作集中在组织特异性果蝇P元件前mRNA外显子剪接沉默元件的表征。我们小组最近的工作集中在RNA结合蛋白PSI和hrp 48的作用。利用这些信息，我们希望确定新的果蝇细胞剪接沉默元件，这两个剪接因子控制。PSI蛋白还与U1 snRNP相互作用，并且消除这种相互作用的PSI突变果蝇株表现出雄性求偶行为缺陷和果蝇雄性特异性无结果前mRNA同种型的改变的前mRNA剪接。我们想研究PSI蛋白如何控制无结果的前mRNA剪接，以及它如何控制果蝇转录组上U1 snRNP的结合。U1 snRNP在PCPA中的U1 snRNP结合位点（过早切割和聚腺苷酸化）、内含子5'剪接位点处的剪接和潜在的新剪接沉默子处具有不同的作用。

 DESCRIPTION (provided by applicant): Mobile genetic elements or transposons are found in the genomes of all organisms. These elements can move via DNA or RNA intermediates. About 50% of the human genome is made up of transposable elements with ~ 2.7% corresponding to DNA-based transposons. Many of these putative transposons or transposase-related genes are uncharacterized. Our previous studies have focused on the P element family of DNA transposons in Drosophila. P element transposase functions as a tetramer, using GTP as a cofactor for transposition. N-terminal domain of the transposase corresponds to a C2CH THAP DNA binding domain, which is a member of a prevalent family of DNA binding domains found exclusively in animal genomes. One THAP gene, called THAP9, is homologous to the Drosophila P element transposase and is present in primates, Xenopus, zebrafish and Ciona, but is absent from rodents. Recent work from our lab has shown that the human and zebrafish THAP9 genes can mobilize the Drosophila and zebrafish P element transposons in human and Drosophila cells. This proposal is focused on understanding what role the human THAP9 gene may play in human embryonic stem cells and how the Drosophila P element transposase protein recognizes and assembles with the transposon ends, donor DNA, target DNA and GTP/Mg2+ to form an active protein-DNA complex. These studies are aimed at gaining mechanistic insights. Alternative pre-mRNA splicing is an important mechanism for regulating gene expression in metazoans and is a conduit through which genomic sequence is transferred to proteomic information. Most eukaryotic genes are split and have the potential for alternative splicing, dramatically increasing proteomic diversity. Many human and mouse disease gene mutations affect the splicing process. Splicing silencers are a major type of RNA control element generating tissue- or cell type-specific alternative splicing patterns. Our previous work has focused on characterization of the tissue-specific Drosophila P element pre-mRNA exonic splicing silencer element. Recent work from our group has focused on how the action of the RNA binding proteins, PSI and hrp48. Using this information, we want to identify new Drosophila cellular splicing silencer elements that are controlled by these two splicing factors. The PSI protein also interacts with U1 snRNP and PSI mutant Drosophila strains that abolish this interaction exhibit male courtship behavior defects and altered pre-mRNA splicing of the Drosophila male-specific fruitless pre-mRNA isoforms. We want to investigate how the PSI protein controls fruitless pre-mRNA splicing and how it controls binding of U1 snRNP on the Drosophila transcriptome. U1 snRNP has distinct roles in U1 snRNP binding sites in PCPA (premature cleavage and polyadenylation), splicing at intron 5' splice sites and at potential new splicing silencers.