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DNA transposons and alternative pre-mRNA splicing.

DNA 转座子和选择性前 mRNA 剪接。

基本信息

批准号：
10630834
负责人：
DONALD C RIO
金额：
$ 70.98万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-15 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10630834
关键词：
5&apos Splice Site Affect Alternative Splicing Amyotrophic Lateral Sclerosis Animals Behavior Binding Binding Sites Biological Markers Cells Complex Courtship Cryoelectron Microscopy DNA DNA Binding Domain DNA Transposable Elements DNA Transposons Defect Disease Drosophila genus Elements Exhibits Family Gene Expression Gene Mutation Genes Genome Genomics Guanosine Triphosphate Health Human Human Genome Introns Link Malignant Neoplasms Mobile Genetic Elements Mus N-terminal Neurodegenerative Disorders Neurons Organism Pathway interactions Pattern Play Poly A Polyadenylation Primates Process Protein Isoforms Protein Structure Initiative Proteins Proteomics RNA RNA Binding RNA Splicing RNA-Binding Proteins Reaction Rodent Role Somatic Mutation Spliceosomes Split Genes Structure Tissues Transcriptional Silencer Elements Transposase U1 Small Nuclear Ribonucleoprotein United States National Institutes of Health Work Xenopus Zebrafish cell type cofactor human disease human embryonic stem cell insight mRNA Precursor male member mutant premature transcriptome

项目摘要

NIH R35 GM118121; DNA transposons and alternative pre-mRNA splicing. D. Rio – PI. PROJECT SUMMARY / ABSTRACT DNA transposons and alternative pre-mRNA splicing. D. Rio – PI 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. We have also used cryo-EM to solve the structure of the P element transposase strand transfer complex. This proposal is focused on understanding what role the human THAP9 gene may play in human embryonic stem cells and the reaction pathway that the Drosophila P element transposase protein uses to recognize and assemble 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. in fact, somatic mutations in splicing factor and spliceosomal genes have been linked to human diseases, such as cancer and the neurodegenerative disease amyotrophic lateral sclerosis (ALS). 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 and the human RNA binding splicing factors hnRNPA1 and DDX5. We are using this information to identify new Drosophila cellular splicing silencer elements that are controlled by PSI and hrp48. We are also analyzing mutant forms of hnRNPA1 that are linked to ALS to find splicing pattern defects that could be used as biomarkers for the disease or provide clues to have neurons are dying in the disease. Splicing silencers are a major type of RNA control element generating tissue- or cell type-specific alternative splicing patterns. 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, at cryptic 5' splice sites and at splicing silencers (from our work).

美国国立卫生研究院 R35 GM118121； DNA 转座子和选择性前 mRNA 剪接。 D. Rio – PI。项目概要/摘要 DNA 转座子和选择性前 mRNA 剪接。 D. Rio – PI 在所有生物体的基因组中都发现了移动遗传元件或转座子。这些元素可以通过 DNA 或 RNA 中间体移动。人类基因组的大约 50% 由转座元件约 2.7% 对应于基于 DNA 的转座子。其中许多推定的转座子或转座酶相关基因尚未表征。我们之前的研究有重点关注果蝇 DNA 转座子的 P 元件家族。 P元件转座酶功能作为四聚体，使用 GTP 作为转座的辅助因子。转座酶的 N 端结构域对应于 C2CH THAP DNA 结合域，它是常见 DNA 家族的成员仅在动物基因组中发现的结合域。一种称为 THAP9 的 THAP 基因是与果蝇 P 元件转座酶同源，存在于灵长类动物、非洲爪蟾、斑马鱼中和 Ciona，但啮齿动物中不存在。我们实验室最近的工作表明，人类和斑马鱼THAP9基因可以调动人类果蝇和斑马鱼P元件转座子和果蝇细胞。我们还使用冷冻电镜来解析 P 元素的结构转座酶链转移复合物。该提案的重点是了解人类的角色 THAP9基因可能在人胚胎干细胞中发挥的作用及果蝇的反应途径 P元件转座酶蛋白用于识别并与转座子末端、供体进行组装 DNA、靶标 DNA 和 GTP/Mg2+ 形成活性蛋白质-DNA 复合物。这些研究旨在获得机械的见解。选择性前mRNA剪接是调节基因表达的重要机制后生动物，是基因组序列转移到蛋白质组信息的管道。大多数真核基因都是分裂的，并且具有选择性剪接的潜力，从而显着增加蛋白质组多样性。许多人类和小鼠疾病基因突变都会影响剪接过程。在事实上，剪接因子和剪接体基因的体细胞突变已与人类疾病，例如癌症和神经退行性疾病肌萎缩侧索硬化症 (ALS)。我们之前的工作重点是组织特异性果蝇 P 元件预表征 mRNA 外显子剪接沉默元件。我们小组最近的工作重点是如何采取行动 RNA 结合蛋白 PSI 和 hrp48 以及人类 RNA 结合剪接因子 hnRNPA1 和DDX5。我们正在利用这些信息来识别新的果蝇细胞剪接沉默元件由 PSI 和 hrp48 控制。我们还分析了 hnRNPA1 的突变形式与 ALS 相关联以发现剪接模式缺陷，这些缺陷可用作该疾病的生物标志物或提供神经元在疾病中死亡的线索。剪接沉默子是RNA的主要类型控制元件产生组织或细胞类型特异性的选择性剪接模式。 PSI蛋白还与 U1 snRNP 和 PSI 突变果蝇菌株相互作用，消除了这种相互作用雄性求爱行为缺陷和果蝇雄性特异性的前mRNA剪接改变无果的前 mRNA 亚型。我们想研究 PSI 蛋白如何控制无结果的前 mRNA 剪接及其如何控制 U1 snRNP 在果蝇转录组上的结合。 U1 snRNP 有 U1 snRNP 结合位点在 PCPA（过早切割和多聚腺苷酸化）、剪接中的不同作用在内含子 5' 剪接位点、隐秘 5' 剪接位点和剪接消音器处（来自我们的工作）。