Biogenesis of macromolecular machines for post-transcriptional regulation of translation

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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.