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

项目摘要 真核基因的表达依赖于细胞核和细胞质中的许多步骤。在 在细胞核中，产生大的新生RNA，如核糖体RNA和mRNA， 组装成核糖核蛋白颗粒（RNP），这些颗粒被输出到细胞质中， 合成.参与RNP组装和输出的蛋白质在整个基因组中起着关键作用， 从共转录RNA加工到翻译的表达。 AAA+ ATP酶是一个功能多样的蛋白质大家族，其利用蛋白质的能量， ATP结合和水解诱导各种蛋白质的构象变化和重构 印刷受体. Elf 1（延伸样因子1）的缺失，一种AAA+超家族ATP酶，参与 RNA核输出，导致严重的生长缺陷，可以通过自发的 抑制基因突变 我们确认并分离了两个抑制突变体：一个内切核酸酶Cue 2和一个大的 核糖体亚基蛋白Rp 12702。Elf 1与这些突变体共同纯化，提供额外的 支持其功能连接。利用亲和纯化和质谱 分析Elf 1和Cue 2，我们已经开发了一个分子框架，系统地 研究它们在转录后基因表达的多方面调控中的作用， RNP输出到翻译核糖体相关的质量控制。此外，我们还观察到， RNA输出缺陷伴Elf 1缺失。由此产生的RNA的核滞留使细胞不稳定， 这可能是因为异常的DNA-RNA杂交（R环）的形成。 我们假设Elf 1与RNP相关，并在RNA/核糖体输出中起作用 和翻译在不同的细胞隔室，拮抗调节的Cue 2。测试 基于这一假设，我们将研究Elf 1、Cue 2和Rp 12702在 维持基因组稳定性（Aim 1），RNA和/或核糖体核输出（Aim 2），和 翻译延伸和核糖体相关的质量控制（目标3）。我们将使用传统的 分子生物学和生物化学方法，并开发新的遗传工具来分析 转录依赖的超重组，并检查各种类型的核糖体- 相关mRNA衰变。我们的假设和研究策略是基于一系列的 初步调查结果。研究结果有望推动RNA生物学、蛋白质 合成和基因组不稳定性。通过研究和学生培训的实施，我们 将产生新的定量遗传工具， 社区

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.