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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).

NIH R35 GM118121；DNA转座子和前mRNA选择性剪接。D.里约热内卢-派。项目摘要/摘要 DNA转座子和可选择的前-mRNA剪接。D.里约热内卢-派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中间体移动。大约50%的人类基因组由转座元件，约2.7%对应于基于DNA的转座子。其中许多推测的转座子或转座酶相关基因尚未确定。我们之前的研究已经重点研究了果蝇DNA转座子的P元件家族。P元素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-末端结构域对应于C2CH Thap DNA结合域，它是流行的DNA家族的成员仅在动物基因组中发现的结合域。一种名为THAP9的thap基因是与果蝇P元素转座酶同源，存在于灵长类动物非洲爪哇、斑马鱼中和Ciona，但在啮齿动物中不存在。我们实验室最近的研究表明，人类和斑马鱼THAP9基因对果蝇和斑马鱼P元件转座子的动员作用和果蝇细胞。我们还用低温电子显微镜计算了P元素的结构转座酶链转移复合体。这项建议的重点是了解人类的角色是什么 THAP9基因可能在人胚胎干细胞和果蝇的反应途径中发挥作用 P元件转座酶蛋白用于识别和组装转座子末端，供体 DNA、靶DNA和GTP/Mg2+形成具有活性的蛋白质-DNA复合体。这些研究的目的是获得机械的洞察力。选择性Pre-mRNA剪接是调控基因表达的重要机制后生动物，是基因组序列传递到蛋白质组信息的管道。大多数真核基因是分裂的，具有选择性剪接的潜力，显著增加了蛋白质组多样性。许多人类和老鼠疾病的基因突变会影响剪接过程。在……里面事实上，剪接因子和剪接体基因的体细胞突变与人类有关癌症和神经退行性疾病肌萎缩侧索硬化症(ALS)。我们之前的工作主要集中在组织特异性果蝇P元件Pre-2的表征上。信使核糖核酸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在RNA结合蛋白中，PSI和hrp48以及人RNA结合剪接因子hnRNPA1 和DDX5。我们正在利用这一信息来鉴定新的果蝇细胞剪接沉默元件由PSI和hrp48控制。我们还在分析hnRNPA1的突变形式，这些突变形式连接到肌萎缩侧索硬化症，以发现剪接模式缺陷，可用作疾病的生物标记物或为神经元在疾病中死亡提供线索。剪接沉默是RNA的一种主要类型控制元件产生特定于组织或细胞类型的选择性剪接图案。PSI蛋白也与取消这种相互作用的U1 SnRNP和PSI突变果蝇菌株相互作用果蝇雄性特有的雄性求偶行为缺陷和改变的Pre-mRNA剪接无果的前信使核糖核酸亚型。我们想要研究PSI蛋白是如何控制无果的Pre-mRNA的剪接及其如何控制U1 SNRNP与果蝇转录组的结合。U1 SnRNP具有在PCPA(过早切割和多腺苷基化)、剪接中U1 SnRNP结合位点的不同作用在内含子5‘剪接点，在神秘的5’剪接点和在剪接消音器(来自我们的工作)。