DNA transposons and alternative pre-mRNA splicing.

DNA 转座子和选择性前 mRNA 剪接。

• 批准号: 10429905
• 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
• 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/10429905
• 关键词: 5' 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; Phosphorus; Play; Polyadenylation; Primates; Process; Protein Isoforms; Protein Structure Initiative; Proteins; Proteomics; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Reaction; Rodent; Role; Somatic Mutation; Split Genes; Structure; Tissues; Transcriptional Silencer Elements; Transposase; U1 Small Nuclear Ribonucleoprotein; United States National Institutes of Health; Work; Xenopus; Zebrafish; base; cell type; cofactor; human disease; human embryonic stem cell; insight; mRNA Precursor; male; member; mutant; premature; transcriptome

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).

NIH R35 GM118121; DNA转座子和替代前MRNA剪接。 D. Rio - Pi。 项目摘要 /摘要 DNA转座子和替代前MRNA剪接。 D. Rio - Pi 在所有生物的基因组中都发现了移动遗传元素或转座子。这些 元素可以通过DNA或RNA中间体移动。大约50％的人类基因组由 约2.7％的转座元素对应于基于DNA的转座子。其中许多 推定的转座子或转座酶相关基因未经表征。我们以前的研究有 专注于果蝇中DNA转座子的P元素家族。 P元素转座酶功能 作为四聚体，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结合域，该结构域是普遍的DNA家族的成员 在动物基因组中仅发现的结合结构域。一个称为thap9的基因是 与果蝇P元件转座酶同源，并存在于私人，Xenopus，斑马鱼中 和ciona，但啮齿动物不存在。我们实验室的最新工作表明，人类和 斑马鱼Thap9基因可以动员人类的果蝇和斑马鱼P元件转座子 和果蝇细胞。我们还使用冷冻EM来解决P元素的结构 转座酶链转移复合物。该提议的重点是理解人类的作用 thap9基因可能在人类胚胎干细胞和果蝇的反应途径中发挥作用 P元素转座酶蛋白用来识别并与转子末端识别和组装供体 DNA，靶DNA和GTP/MG2+形成活性蛋白-DNA复合物。这些研究针对 获得机械洞察力。 替代前MRNA剪接是调节基因表达的重要机制 后生动物是一个导管，将基因组序列转移到蛋白质组学信息中。 大多数真核基因分裂，并且具有替代分裂的潜力，大幅增加 蛋白质组学多样性。许多人类和小鼠疾病基因突变会影响剪接过程。 事实，剪接因子和剪接基因中的体细胞突变与人有关 疾病，例如癌症和神经退行性疾病肌营养性侧索硬化症（ALS）。 我们以前的工作重点是表征组织特异性果蝇P元件的表征 mRNA外显子剪接消音器元件。我们小组的最新工作集中于该行动 RNA结合蛋白，PSI和HRP48以及人RNA结合剪接因子HNRNPA1 和DDX5。我们正在使用此信息来识别新的果蝇细胞剪接消音器元素 由PSI和HRP48控制。我们还正在分析HNRNPA1的突变形式 链接到ALS，以找到可以用作疾病的生物标志物或 提供线索使神经元死于该疾病。剪接硅是主要的RNA 控制元件生成组织或细胞类型特异性替代剪接模式。 PSI蛋白 还与废除这种相互作用的U1 SNRNP和PSI突变果蝇菌株相互作用 果蝇特异 毫无结果的前MRNA同工型。我们想研究PSI蛋白如何控制毫无结果的前MRNA 剪接及其如何控制U1 SNRNP在果蝇转录组上的结合。 U1 SNRNP具有 在PCPA（过早切割和聚腺苷酸化）中的U1 SNRNP结合位点中的不同作用，剪接 在内含子5'剪接站点，在加密5'剪接站点和剪接消音器（从我们的工作中）处。