Biogenesis of macromolecular machines for post-transcriptional regulation of translation

用于翻译转录后调控的大分子机器的生物发生

• 批准号: 10454992
• 负责人: Homa Ghalei
• 金额: $ 37.96万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-17 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10454992
• 关键词: Biochemical; Biogenesis; Biological Assay; Cells; Complex; Data; Defect; Deposition; Disease; Eukaryota; Event; Gene Expression; Genetic; Genetic Transcription; Goals; Laboratories; Lead; Mediating; Methylation; Modification; Molecular; Mutation; Nerve Degeneration; Nucleotides; Pathway interactions; Pattern; Perception; Play; Post-Transcriptional Regulation; Process; Production; Protein Biosynthesis; Proteins; Proteomics; Public Health; RNA; Reagent; Regulation; Ribonucleoproteins; Ribosomal RNA; Ribosomes; Role; Site; Small Nucleolar RNA; Small Nucleolar Ribonucleoproteins; Textbooks; Therapeutic Intervention; Translations; Untranslated RNA; Yeasts; cancer type; cell growth; human disease; insight; new therapeutic target; next generation sequencing; novel; protein complex; structural biology; tool

Summary: Ribosomes are highly conserved RNA-protein complexes that direct protein synthesis in all cells. Dysregulation of ribosome production or function is detrimental to gene expression and underlies several disease states. Over 2% of ribosomal RNA (rRNA) nucleotides are modified. These modifications play a critical role in the proper production of ribosomes that can accurately perform protein synthesis. The two major rRNA modifications are 2’-O-methylation and pseudouridylation that are directed by highly conserved non-coding RNAs called small nucleolar RNAs (snoRNAs). Altered levels of snoRNAs are associated with human diseases from neurodegeneration to multiple types of cancer, underscoring their importance for proper cell growth. Therefore, a key question is how levels of snoRNAs are regulated and how does their dysregulation lead to translation defects in disease? Despite the textbook perception that rRNA modifications are equally deposited in all ribosomes, recent advances in mapping modifications have revealed substoichiometric rRNA modification sites, strongly suggesting that ribosome assembly and function may be regulated by the modification status of rRNA. A long-term goal of my laboratory is to identify the post-transcriptional mechanisms that regulate the abundance of snoRNAs and understand their contribution to cellular translational control. A prominent rRNA modification in eukaryotes is 2’-O-methylation, the incorporation of which is guided by snoRNAs of the box C/D class. These snoRNAs interact with a set of evolutionarily conserved proteins to form ribonucleoprotein complexes (snoRNPs). The assembly of snoRNPs is highly regulated which, in turn, is important to maintain levels of snoRNAs and to coordinate this process with other cellular events. However, despite their fundamental importance, much of these regulatory events remains a black box. We have performed targeted yeast mutational and suppressor screens of snoRNP assembly factors to determine their essential contributions and identify genetic pathways that mediate snoRNP biogenesis. Our data indicate that regulation of box C/D snoRNP production by assembly factors is critically important for control of the modification pattern of rRNAs and dysregulation of this process alters the biogenesis pathway and the fidelity of ribosomes. Our goal is to combine the novel genetic tools and reagents that we have recently developed with biochemical assays, structural biology, proteomics, and next-generation sequencing to answer two key questions: 1) How do regulatory factors control the steady-state levels of snoRNAs required for accurate modification of rRNA?; and 2) How do changes in snoRNA levels alter and tune protein synthesis? These studies will provide significant insights into the control of gene expression by snoRNAs at the translation level, and may inform our view of how snoRNA dysregulation underlies human disease.

概括： 核糖体是高度保守的 RNA-蛋白质复合物，指导所有细胞中的蛋白质合成。失调 核糖体产生或功能的紊乱不利于基因表达，并且是多种疾病状态的基础。超过 2% 的核糖体 RNA (rRNA) 核苷酸被修饰。这些修改在适当的情况下发挥着关键作用 生产可以准确进行蛋白质合成的核糖体。两个主要的 rRNA 修饰是 2'-O-甲基化和假尿苷化由高度保守的非编码 RNA（称为小RNA）引导 核仁 RNA (snoRNA)。 snoRNA 水平的改变与人类疾病有关 神经变性导致多种类型的癌症，强调了它们对正常细胞生长的重要性。所以， 一个关键问题是 snoRNA 的水平如何调节以及它们的失调如何导致翻译 疾病缺陷？尽管教科书认为 rRNA 修饰同等地沉积在所有 核糖体，作图修饰的最新进展揭示了亚化学计量的 rRNA 修饰位点， 强烈表明核糖体的组装和功能可能受到rRNA修饰状态的调节。 我实验室的一个长期目标是确定调节丰度的转录后机制 snoRNA 并了解它们对细胞翻译控制的贡献。一个显着的 rRNA 修饰 真核生物是 2’-O-甲基化，其掺入是由盒 C/D 类的 snoRNA 引导的。这些 snoRNA 与一组进化上保守的蛋白质相互作用形成核糖核蛋白复合物 （snoRNP）。 snoRNP 的组装受到高度调控，这对于维持 snoRNP 的水平非常重要 snoRNA 并协调该过程与其他细胞事件。然而，尽管它们的基本 重要的是，这些监管事件中的大部分仍然是一个黑匣子。我们进行了定向酵母突变 和抑制筛选snoRNP组装因子以确定其基本贡献并识别 介导 snoRNP 生物发生的遗传途径。我们的数据表明框 C/D snoRNP 的调节 组装因子的产生对于控制 rRNA 的修饰模式至关重要 这一过程的失调会改变生物发生途径和核糖体的保真度。我们的目标是结合 我们最近通过生化分析、结构生物学开发的新型遗传工具和试剂， 蛋白质组学和下一代测序回答两个关键问题：1）调控因素如何控制 精确修饰 rRNA 所需的 snoRNA 的稳态水平？ 2）如何改变 snoRNA 水平改变和调节蛋白质合成？这些研究将为控制 snoRNA 在翻译水平上的基因表达，并可能告诉我们 snoRNA 失调如何发生的观点 是人类疾病的基础。