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

• 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
• 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

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.

项目摘要 真核基因表达取决于细胞核和细胞质的许多步骤。在 核，大新生RNA，例如核糖体RNA和mRNA，是制造的，并且 聚集成核糖核蛋白颗粒（RNP），这些颗粒被导出到细胞质中的蛋白质 合成。 RNP组装和出口涉及的蛋白质在整个基因中起关键作用 从共转录RNA处理到翻译的表达。 AAA+ ATPases是一个大型且功能多样的蛋白质家族，它使用的能量 ATP结合和水解以诱导各种蛋白质的构象变化和重塑 基材。 ELF1的损失（延伸样因子1），AAA+超家族ATPase涉及 RNA核出口，导致严重的生长缺陷，可以自发缓解 抑制突变。 我们确认并隔离了两个抑制剂突变体：一个核酸内切酶，CUE2和一个大的 核糖体亚基蛋白，RPL2702。 ELF1与这些突变体共同固定，提供了更多 支持其功能连接。使用亲和力纯化和质谱法 ELF1和CUE2的分析，我们已经开发了一个分子框架 研究它们在转录后基因表达的多方面调节中的作用 RNP导出为核糖体相关质量控制。另外，我们已经观察到 RNA导出缺陷，损失ELF1。由此产生的RNA的核保留破坏了 基因组，可能是因为异常的DNA-RNA杂种（R-loops）形式。 我们假设ELF1与RNP和RNA/核糖体导出中的功能有关 和不同的细胞室中的翻译，由CUE2拮抗。测试 这个假设，我们将研究ELF1，CUE2和RPL2702在 保持基因组稳定性（AIM 1），RNA和/或核糖体核出口（AIM 2），以及 翻译伸长和与核糖体相关的质量控制（AIM 3）。我们将使用传统 分子生物学和生化方法，还开发了新的遗传工具来分析 转录依赖性超级聚集并检查各种类型的核糖体 相关的mRNA衰变。我们的假设和研究策略是基于许多 初步发现。预计结果将推进RNA生物学，蛋白质的领域 合成和基因组不稳定性。通过实施研究和学生培训，我们 将生成新的定量遗传工具，这些工具将在裂变酵母中得到理解 社区。