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RNA processing in Drosophila development

果蝇发育中的RNA加工

基本信息

批准号：
9154664
负责人：
Ruth M STEWARD
金额：
$ 31万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
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：
2016-09-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9154664
关键词：
Affect Alleles Binding Binding Proteins Binding Sites Biochemical Genetics Blood Vessels Brain Cancer Cell Growth Cardiac Cell Maintenance Cell Nucleus Cell division Cells Chromosomes Collaborations Complex Cytoplasm DNA DNA Binding DNA Methylation Data Defect Development Diagnostic Disease Drosophila genus Embryo Employee Strikes Epigenetic Process Gene Expression Genes Genetic Transcription Hematopoietic Hematopoietic stem cells Human Immunohistochemistry Individual Infertility Light Link Location Maintenance Malignant Neoplasms Maps Messenger RNA Metabolism Methods Mixed Function Oxygenases Modification Molecular Molecular Genetics Mus Neurologic Neurons Nuclear Export Ovarian Ovary Phenotype Polyribosomes Post-Transcriptional Regulation Process Proteins RNA RNA Interference RNA Processing RNA Splicing Recruitment Activity Regenerative Medicine Retinal Ribosomes Role Site Stem cells Testing Tetanus Helper Peptide Tissues Transcript Translation Process Translational Repression Translations Universities Vertebrates abstracting base cancer therapy demethylation fly knock-down leukemia mutant neurodevelopment neuroepithelium novel protein function relating to nervous system research study ribosome profiling stem cell differentiation transcriptome

项目摘要

Abstract The aim of our proposal is to elucidate the mechanisms that link transcription of specific RNAs in the nucleus to their translation (RNA processing). We have identified and characterized a novel and highly conserved gene, Zfrp8/PDCD2, and shown that it is essential in stem cells in flies, mouse, and human, and that it is also required for growth of cancer cells. We discovered Zfrp8/PDCD2 is required for the nuclear export of select mRNAs and TE transcripts. Also it interacts with the small ribosomal subunit and forms a complex with mRNA binding proteins. Our data suggest that Zfrp8/PDCD2 controls subcellular localization of select RNAs and the association of mRNARNPs with ribosomes. We also have identified Tet/TET1 as a new Zfrp8/PDCD2 interacting protein. In vertebrates, TET proteins function in DNA demethylation converting 5methylcytosine (5mC) into 5hydroxymethylcytosine (5hmC), modifications that are not detected in Drosophila DNA. A recent study shows that vertebrate Tet proteins can also convert 5mrC to 5hmrC in RNA. Inspired by this discovery, we have shown that 5hmrC also exists in flies and depends on Tet activity. We hypothesize that Tet modifies specific transcripts and regulates the recruitment of Zfrp8 to these RNAs, so controlling their processing and translation. We propose to test this hypothesis by a combination of molecular/biochemical and genetic experiments. We will identify 5hmrCmodified transcripts transcriptomewide and study their integrity, levels, and localization in wild type and mutant tissues. We will map the Tet binding sites on DNA and compare their location to 5hmrCmodified transcripts. Finally, we will test how Tet and Zfrp8 affect ribosomal occupancy of mRNAs and establish how Tet and 5hmrC affect their translation. In addition to investigating the mechanism by which Tet and 5hmrC regulate RNA metabolism and how Zfrp8 affects the process, we will determine the importance of both genes in development and stem cell differentiation by studying the mutant phenotypes of new Tet alleles and Tet and Zfrp8 KD tissues. Because of the conservation of both Tet and Zfrp8/PDCD2 our results are likely to shed light on the same process in mouse and human.

摘要我们的建议的目的是阐明连接特定RNA转录的机制，我们已经鉴定并表征了一种新的高度特异性的保守基因Zfrp 8/PDCD 2，并表明它在苍蝇，小鼠和人类的干细胞中是必需的，我们发现Zfrp 8/PDCD 2是细胞核输出所必需的，选择mRNA和TE转录本。它还与核糖体小亚基相互作用，形成复合物，我们的数据表明，Zfrp 8/PDCD 2控制选择性细胞因子的亚细胞定位。我们还鉴定了泰特/TET 1作为一种新的转录因子，在脊椎动物中，泰特蛋白在DNA去甲基化转化中起作用， 5 ′甲基胞嘧啶（5 mC）转化为5 ′羟甲基胞嘧啶（5 hmC），这些修饰在最近的一项研究表明，脊椎动物Tet蛋白也可以将RNA中的5 mrC转化为5 hmrC。受这一发现的启发，我们发现5 hmrC也存在于果蝇中，并依赖于泰特的活性。假设泰特修饰特定的转录物并调节Zfrp 8向这些RNA的募集，控制它们的处理和翻译。我们建议通过分子/生物化学和遗传学的结合来验证这一假设。我们将在转录组范围内鉴定5 hmrC β修饰的转录物，并研究它们的完整性，水平，我们将绘制泰特在DNA上的结合位点，并比较它们在野生型和突变型组织中的定位。最后，我们将测试泰特和Zfrp 8如何影响核糖体占有率。 mRNA，并建立如何泰特和5 hmrC影响他们的翻译。泰特和5 hmrC调节RNA代谢以及Z frp 8如何影响这一过程，我们将确定这两个基因在发育和干细胞分化中的重要性，新的泰特等位基因和泰特和Zfrp 8 KD组织。由于泰特和Zfrp 8/PDCD 2的保守性，这些结果很可能揭示小鼠和人类的相同过程。