Uncovering the Functions of Expansion Segments

Abstract

Ribosomes are primordial cellular components which mediate the production of proteins by translating mRNA into polypeptide chains. The core catalytic sites of ribosomes consist of ribosomal RNA (rRNA) and are conserved across both eukaryotic and prokaryotic species. However, eukaryotic rRNAs are distinguished by the presence of expansion segments (ESs), surface-exposed stretches of RNA present at various sites throughout their secondary structure, comprising up to a third of the mature rRNA sequence. While several expansion segments in single-celled eukaryotes have been shown to play a role in mediating translation and ribosome biogenesis, the functional properties of metazoan expansion segments, which are much longer and more structurally complex, remain largely undefined due to technical intractability. Using novel biochemical and recombinant ribosomal DNA (rDNA) approaches, we developed an experimental framework to investigate the functions of metazoan expansion segments, using ES39La as a case study. We used mutant recombinant rDNA in which ES39La has been deleted, and found that the deletion has minimal, but measurable, effects on ribosome biogenesis and global translation. We also began developing an approach for ribosome profiling of mutant recombinant ribosomes to examine the possible function of expansion segments in translating specific mRNAs. In parallel, we used novel biochemical methods to isolate ES39La-binding proteins, such that their roles in translation and ribosome biogenesis can be characterized. Together, the completion of aims in this study will provide a blueprint to facilitate the elucidation of other expansion segment regions in the future.