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Investigation of multifunctional proteins that integrate packaging RNPs, RNA export, and translation

研究整合包装 RNP、RNA 输出和翻译的多功能蛋白质

基本信息

批准号：
10047135
负责人：
Ke Zhang Reid
金额：
$ 43.01万
依托单位：
WAKE FOREST UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10047135
关键词：
ATP phosphohydrolase Affinity Affinity Chromatography Binding Biochemical Biological Assay Biological Process Biology Cell Nucleus Cell physiology Cells Chemistry Communities Complex Cytoplasm DNA Data Defect Diamond Diamond-Blackfan anemia Dyskeratosis Congenita Environment Etiology Family Fission Yeast Gene Expression Genetic Genetic Recombination Genetic Transcription Genetic Translation Genome Genome Stability Genomic Instability Growth Hybrids Hydrolysis Intelligence Investigation Label Lead Malignant Neoplasms Maps Mass Spectrum Analysis Measures Mediating Messenger RNA Molecular Molecular Biology Molecular Conformation Mutation Nature Nuclear Nuclear Export Nuclear RNA Phenotype Play Post-Transcriptional Regulation Process Protein Biosynthesis Protein Family Protein Subunits Proteins Quality Control Quantitative Genetics RNA RNA Processing Research Research Training Ribonucleoproteins Ribosomal Proteins Ribosomal RNA Ribosomes Role Saccharomycetales Suppressor Mutations Symptoms Syndrome System Testing Transcription Elongation Translations Universities Work base career college doctoral student endonuclease fluorescence imaging forest human disease implementation research innovation mRNA Decay messenger ribonucleoprotein mutant paralogous gene particle polysome profiling prevent ribosome profiling student training tool trafficking

项目摘要

Project Summary Eukaryotic gene expression depends on many steps in both the nucleus and cytoplasm. In the nucleus, large nascent RNAs, such as ribosomal RNAs and mRNAs, are manufactured and assembled into ribonucleoprotein particles (RNPs) that are exported to the cytoplasm for protein synthesis. The proteins involved in RNP assembly and export play critical roles throughout gene expression from co-transcriptional RNA processing to translation. AAA+ ATPases are a large and functionally diverse family of proteins that use the energy of ATP binding and hydrolysis to induce conformational changes and remodeling in various protein substrates. Loss of Elf1 (Elongation-Like Factor 1), an AAA+ superfamily ATPase implicated in RNA nuclear export, causes severe growth defects that can be mitigated by spontaneous suppressor mutations. We confirmed and isolated two suppressor mutants: an endonuclease, Cue2, and a large ribosomal subunit protein, Rpl2702. Elf1 co-purifies with these mutants, providing additional support for their functional connection. Using affinity purification and mass spectrometry analysis of Elf1 and Cue2, we have developed a molecular framework to systematically investigate their roles in the multifaceted regulation of posttranscriptional gene expression from RNP export to translation to ribosome-associated quality control. In addition, we have observed RNA export defects with loss of Elf1. The resulting nuclear retention of RNA destabilizes the genome, probably because abnormal DNA-RNA hybrids (R-loops) form. We hypothesize that Elf1 is associated with RNPs and functions in RNA/ribosome export and translation in different cellular compartments, antagonistically regulated by Cue2. To test this hypothesis, we will investigate the integrated roles of Elf1, Cue2, and Rpl2702 in maintaining genome stability (Aim 1), RNA and/or ribosome nuclear export (Aim 2), and translation elongation and ribosome-associated quality control (Aim 3). We will use traditional molecular biology and biochemical approaches, and also develop new genetic tools to analyze transcription-dependent hyper-recombination and examine various types of ribosome- associated mRNA decays. Our hypotheses and research strategy are based on a host of preliminary findings. Results are expected to advance the fields of RNA biology, protein synthesis, and genomic instability. Through implementation of research and student training, we will generate new quantitative genetic tools that will be appreciated in the fission yeast community.

项目总结