Biogenesis of macromolecular machines for post-transcriptional regulation of translation

摘要： 核糖体是高度保守的RNA-蛋白质复合体，指导所有细胞中蛋白质的合成。调控失调 核糖体生产或功能的缺失不利于基因表达，是几种疾病状态的基础。完毕 2%的核糖体RNA(RRNA)核苷酸被修饰。这些修改在适当的 生产能够准确地进行蛋白质合成的核糖体。两个主要的rRNA修饰是 由高度保守的非编码RNA引导的2‘-O-甲基化和假尿酸 核仁RNA(SnoRNAs)。SnoRNAs水平的改变与人类疾病有关 神经退行性变为多种类型的癌症，强调了它们对细胞正常生长的重要性。因此， 一个关键问题是snoRNAs的水平是如何调节的，它们的失调是如何导致翻译的 疾病中的缺陷？尽管教科书上认为rRNA修饰在所有 核糖体，作图修饰的最新进展揭示了亚化学计量比rRNA修饰位点， 强烈提示核糖体的组装和功能可能受rRNA修饰状态的调控。 我的实验室的一个长期目标是识别调节丰度的转录后机制 并了解它们对细胞翻译控制的贡献。一种显著的rRNA修饰 真核生物是2‘-O-甲基化，其掺入是由盒C/D类的snoRNAs引导的。这些 SnoRNAs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体 (SnoRNPs)。SnoRNP的组装受到严格的调控，这反过来对维持snoRNP的水平非常重要 SnoRNAs，并与其他细胞事件协调这一过程。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。我们已经进行了有针对性的酵母突变 和抑制物筛选snoRNP组装因子以确定它们的基本作用并识别 介导snoRNP生物发生的遗传途径。我们的数据表明，box C/D snoRNP的调节 组装因子的产生对于控制rRNA和 这一过程的失调改变了生物发生途径和核糖体的保真度。我们的目标是联合 我们最近开发的新型遗传工具和试剂，包括生化分析、结构生物学、 蛋白质组学和下一代测序回答两个关键问题：1)调控因子如何控制 准确修饰rRNA所需的snoRNAs的稳定水平？；以及2)在 SnoRNA水平改变和调节蛋白质合成？这些研究将为控制艾滋病提供重要的见解。 SnoRNA在翻译水平上的基因表达，并可能为我们提供关于snoRNA如何失调的观点 是人类疾病的基础。

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.