The role of rRNA modifications in regulation of translation initiation

Emory University, Atlanta GA

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT: Ribosomes produce all cellular proteins through the process of translation. Translation is highly regulated at three major steps: initiation, elongation, and termination, with initiation being the rate-limited and most regulated step. Regulation of translation is vital for cell survival and homeostasis in all living organisms, and dysregulated translation can cause human diseases ranging from cancer to neurological disorders. Ribosomes comprise four ribosomal RNAs (rRNAs) and 80 ribosomal proteins (RPs) in two subunits. rRNAs are chemically modified at >200 sites in humans (>100 in budding yeast). An abundant rRNA modification is 2’-O-methylation (Nm). Recent studies have provided evidence that Nms are not static features of the ribosomes. Instead, Nm levels at specific rRNA sites vary across human tissues, cancer cell lines, tumor types, and during mouse brain development. Despite the significance of Nms, the contributions of Nms to translation regulation remain largely unknown to date. Because ribosomes are highly conserved across species, the single-celled eukaryote Saccharomyces cerevisiae (budding yeast) has long served as a model organism for studies of translation regulation. In fact, many of the regulatory steps of translation were first identified in budding yeast. In yeast and other eukaryotes, Nms are guided by a large class of small nucleolar RNAs (snoRNAs), termed C/D snoRNAs. Our lab has shown that Nm levels on rRNAs can be modulated site-specifically by regulating the level of C/D snoRNAs during snoRNA biogenesis. Yeast cells harboring hypomethylated ribosomes have been shown to have a strong growth defect when compared to wildtype yeast and dysregulated Nms have also been shown to change the binding of translation factors to ribosomes. A high copy suppressor screen performed in our lab has identified specific eukaryotic initiation factors (eIFs) that can rescue this growth defect significantly: 1) the ternary complex (TC) composed of the GTPase eIF2, GTP, and Met-tRNAi together with the eIF2-GTPase activating protein, eIF5, responsible for proper start codon recognition; 2) the GTPase eIF5B, which facilitates subunit joining. The binding site of the TC and eIF5B on ribosomes both harbor Nm sites that could influence the interactions between eIFs and the ribosome. Based on these data, I hypothesize that Nms contribute to the binding of TC to the ribosome and aid in eIF5B-mediated ribosomal subunit joining. I will test this hypothesis through two independent and complementary specific aims. In Aim 1, I will conduct in vivo reporter assays and in vitro biochemical experiments with ribosomes from wildtype yeast and yeast lacking individual Nms to understand the impact of Nms on start codon selection and Met-tRNAi binding. In Aim 2, I will use ribosome biochemistry to understand the impact of Nms on eIF5B binding to the ribosome and on subunit joining. Successful completion of the proposed studies will reveal new insights into the regulation of translation by rRNA modifications. In doing so, I will enhance my knowledge and acquire the training needed to become an independent scientific leader.