DNA transposons and alternative pre-mRNA splicing

 描述(申请人提供)：可移动的遗传元件或转座子存在于所有生物体的基因组中。这些元素可以通过DNA或RNA中间体进行移动。人类基因组约50%由转座元件组成，其中约2.7%对应于DNA转座子。这些假定的转座子或转座酶相关基因中的许多都没有确定的特征。我们以前的研究主要集中在果蝇DNA转座子的P元件家族上。P元件转座酶起四聚体的作用，利用GTP作为转座的辅助因子。转座酶的N-末端结构域对应于一个C2CH-Thap DNA结合域，它是一个普遍存在的DNA结合域家族的成员，该家族仅在动物基因组中发现。一种名为THAP9的thap基因与果蝇P元素转座酶同源，存在于灵长类动物、非洲爪哇、斑马鱼和Ciona中，但在啮齿动物中缺失。我们实验室最近的工作表明，人和斑马鱼THAP9基因可以在人和果蝇细胞中动员果蝇和斑马鱼P元件转座子。这一建议的重点是了解人类THAP9基因在人类胚胎干细胞中可能发挥的作用，以及果蝇P元件转座酶蛋白如何识别并组装转座子末端、供体DNA、靶DNA和GTP/Mg2+形成活性蛋白质-DNA复合体。这些研究旨在获得机械论的见解。选择性Pre-mRNA剪接是后生动物基因表达调控的重要机制，也是基因组序列向蛋白质组信息传递的通道。大多数真核基因是分裂的，具有选择性剪接的潜力，大大增加了蛋白质组的多样性。许多人类和老鼠疾病的基因突变会影响剪接过程。剪接沉默是产生特定组织或细胞类型的选择性剪接模式的一种主要类型的RNA控制元件。我们以前的工作主要集中在组织特异性的果蝇P因子前mRNA外显子剪接沉默元件的鉴定上。我们小组最近的工作集中在RNA结合蛋白PSI和hrp48的作用。利用这些信息，我们想要识别受这两个剪接因子控制的新的果蝇细胞剪接沉默元件。PSI蛋白还与U1、SnRNP和PSI突变的果蝇品系相互作用，取消这种相互作用的果蝇品系表现出雄性求偶行为缺陷，并改变了果蝇雄性特异的无果Pre-mRNA亚型的前mRNA剪接。我们想研究PSI蛋白如何控制无果的Pre-mRNA剪接，以及它如何控制U1 SnRNP与果蝇转录组的结合。U1 SnRNP在PCPA的U1 SnRNP结合位点(过早切割和多聚腺苷化)、内含子5‘剪接位点和潜在的新的剪接沉默中具有不同的作用。

 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.