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 核，大的新生RNA，如核糖体RNA和mRNAs，被制造和 组装成核糖核蛋白颗粒(RNPs)，然后输出到细胞质中获取蛋白质 综合。参与RNP组装和输出的蛋白质在整个基因中起着至关重要的作用 从共转录RNA加工到翻译的表达。 AAA+ATPase是一个功能多样的蛋白质大家族，它利用 ATP结合和水解诱导多种蛋白质的构象变化和重塑 底物。AAA+超家族ATPase Elf1(延伸样因子1)的缺失与 RNA核输出，导致严重的生长缺陷，可以通过自发的方式缓解 抑制子突变。 我们确认并分离了两个抑制突变体：一个内切酶Cue2和一个大的 核糖体亚基蛋白，Rpl2702。Elf1与这些突变体共同纯化，提供了额外的 支持它们的功能连接。利用亲和纯化和质谱学 通过对Elf1和Cue2的分析，我们开发了一个分子框架来系统地 研究它们在转录后基因表达的多方面调控中的作用 RNP输出到翻译为核糖体相关的质量控制。此外，我们还观察到 带有Elf1缺失的RNA输出缺陷。由此产生的RNA的核滞留破坏了 基因组，可能是因为形成了异常的DNA-RNA杂交体(R-环)。 我们假设Elf1与RNPs相关，并在RNA/核糖体输出中发挥作用 和翻译在不同的细胞室，拮抗地被Cue2调节。为了测试 在这一假设下，我们将研究Elf1、Cue2和Rpl2702在 维持基因组稳定(目标1)、核糖核酸和/或核糖体核输出(目标2)；以及 翻译延长和核糖体相关质量控制(目标3)。我们将使用传统的 分子生物学和生化方法，并开发新的遗传工具来分析 转录依赖的超重组，并检测各种类型的核糖体- 相关的信使核糖核酸降解。我们的假设和研究策略基于大量 初步调查结果。这一结果有望推动RNA生物学、蛋白质等领域的发展 合成和基因组的不稳定性。通过实施研究和学生培训，我们 将产生新的量化遗传工具，这些工具将在分裂酵母中受到重视 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.