DNA transposons and alternative pre-mRNA splicing.

NIH R35 GM 118121; DNA转座子和选择性前mRNA剪接。D.里约-派。 项目总结/摘要 DNA转座子和选择性前mRNA剪接。D.里约- PI 移动的遗传元件或转座子存在于所有生物的基因组中。这些 元件可以通过DNA或RNA中间体移动。大约50%的人类基因组是由 转座因子约2.7%对应于基于DNA的转座子。许多这些 推定的转座子或转座酶相关基因未被表征。我们以往的研究 主要研究果蝇中的DNA转座子P元件家族。P元件转座酶功能 作为四聚体，使用GTP作为转座的辅因子。转座酶N端结构域 对应于C2CH THAP DNA结合结构域，其是DNA的普遍家族的成员 仅在动物基因组中发现的结合域。一种叫做THAP 9的THAP基因， 与果蝇P元件转座酶同源，存在于灵长类、爪蟾、斑马鱼中 和玻璃海鞘，但没有啮齿动物。我们实验室最近的工作表明，人类和 斑马鱼THAP 9基因可以调动果蝇和斑马鱼P元件转座子在人类中的表达 和果蝇细胞。我们还用冷冻电镜来解决的P元素的结构 转座酶链转移复合物。这一建议的重点是了解人类的作用， THAP 9基因可能在人类胚胎干细胞和果蝇中起作用的反应途径 P元件转座酶蛋白用于识别并组装转座子末端，供体 DNA、靶DNA和GTP/Mg 2+形成活性蛋白-DNA复合物。这些研究旨在 获得机械的洞察力。 前体mRNA选择性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。 大多数真核基因是分裂的，并具有选择性剪接的潜力， 蛋白质组多样性许多人类和小鼠疾病基因突变影响剪接过程。在 事实上，剪接因子和剪接体基因中的体细胞突变与人类 在一些实施方案中，所述药物用于治疗神经退行性疾病，例如癌症和神经退行性疾病肌萎缩性侧索硬化症（ALS）。 我们以前的工作主要集中在组织特异性果蝇P元件的表征， mRNA外显子剪接沉默子元件。我们小组最近的工作集中在如何行动 RNA结合蛋白PSI和hrp 48以及人RNA结合剪接因子hnRNPA 1 DDX5我们正在利用这些信息来确定新的果蝇细胞剪接沉默元件 由PSI和hrp 48控制。我们还分析了hnRNPA 1的突变形式， 与ALS相关，以发现可用作疾病生物标志物的剪接模式缺陷， 提供了神经元死亡的线索剪接沉默子是RNA的主要类型 产生组织或细胞类型特异性可变剪接模式的控制元件。PSI蛋白 也与U1 snRNP和PSI突变果蝇菌株相互作用，消除这种相互作用， 雄性求偶行为缺陷和果蝇雄性特异性 无结果的前mRNA亚型。我们想研究PSI蛋白如何控制无结果的前mRNA 剪接以及它如何控制果蝇转录组上U1 snRNP的结合。U1 snRNP具有 PCPA中U1 snRNP结合位点的不同作用（过早切割和多聚腺苷酸化），剪接 在内含子5'剪接位点，在隐蔽的5'剪接位点和在剪接沉默子（来自我们的工作）。

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).