Biogenesis of macromolecular machines for post-transcriptional regulation of translation

总结： 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 核糖体的产生或功能对基因表达是有害的，并且是几种疾病状态的基础。超过 2%的核糖体RNA（rRNA）核苷酸被修饰。这些修改在适当的 核糖体的生产，可以准确地进行蛋白质合成。两种主要的rRNA修饰是 2 '-O-甲基化和假尿苷化由高度保守的非编码RNA（称为小分子RNA）指导， 核仁RNA（snoRNA）。snoRNA水平的改变与人类疾病有关， 神经退行性变对多种类型的癌症的影响，强调了它们对适当细胞生长的重要性。因此，我们认为， 一个关键的问题是snoRNA的水平是如何调节的，以及它们的失调是如何导致翻译的 疾病的缺陷？尽管教科书认为rRNA修饰在所有细胞中都是平等的 核糖体，最近在定位修饰方面的进展已经揭示了亚化学计量的rRNA修饰位点， 这强烈表明核糖体的组装和功能可能受rRNA修饰状态的调节。 我的实验室的一个长期目标是确定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。一个突出的rRNA修饰， 在真核生物中，甲基化是2 '-O-甲基化，其掺入由盒C/D类的snoRNA指导。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的组装受到高度调节，这反过来对于维持snoRNP的水平是重要的。 snoRNA和协调这一过程与其他细胞事件。尽管他们的基本 重要的是，这些监管事件中的许多仍然是一个黑匣子。我们进行了靶向酵母突变， 和抑制筛选snoRNP组装因子，以确定它们的基本贡献， 介导snoRNP生物发生的遗传途径。我们的数据表明，盒C/D snoRNP的调节 装配因子的产生对于控制rRNA的修饰模式至关重要， 该过程的失调改变了生物发生途径和核糖体的保真度。我们的目标是将联合收割机 我们最近用生化分析，结构生物学， 蛋白质组学和下一代测序来回答两个关键问题：1）调节因子如何控制 精确修饰rRNA所需的snoRNA的稳态水平？和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 基因表达的snoRNA在翻译水平上，并可能告知我们的观点snoRNA失调如何 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.