Exploring the RNA-Binding Profiles of Ribosomal Protein S15 Through In Vitro Selection

Abstract

Cis-regulatory RNA elements are structured regions of an mRNA that regulate the transcription, translational efficiency, or stability of the mRNA. These cis-regulatory RNAs are widely used across all domains of life to modulate gene expression in response to various stimuli. In bacteria, examples of these cis-regulatory RNAs include small RNAs, structured 50-500 nucleotide non-coding RNA that bind to mRNA or protein to alter expression, and riboswitches, which consist of a ligand-binding aptamer domain whose complex tertiary structure selectively responds to specific ligands to regulate downstream gene expression on the transcriptional or translational level. Ribosomal protein expression in bacteria is often controlled using an autogenous cis-regulatory mechanism, in which select ribosomal proteins (r-proteins) bind RNA structures in the 5’-untranslated of their own mRNA to regulate the expression of r-protein operons. Some of these structures, such as the RNA leaders regulating r-proteins L1, L20, and S2, have striking homology and often mimicry between the recognition motifs within their primary binding partner, ribosomal RNA (rRNA), and their secondary binding partner, the structured mRNA leader. Ribosomal protein S15 is a notable exception to this trend, as the five regulatory RNA leaders identified across various bacterial species that respond to S15 are structurally distinct, narrowly distributed to their respective phyla, and often bear little obvious homology to the rRNA. Additionally, inter-species interaction studies have shown that the S15 homologs from these species have specific recognition profiles fori the mRNA regulators, and not all interactions are reciprocal. How RNA regulators arise and are maintained in bacterial genomes is not well understood, and thus we sought to use ribosomal protein S15 as a model to study how differences in the RNA-binding profiles of the various S15 homologs may have driven the diversity of the mRNA regulators we see today. To explore these RNA-binding profiles, I utilized an in vitro selection approach to enrich for aptamers (structured RNAs that bind a specific ligand) that bind the S15 homologs from Escherichia coli (EcS15), Geobacillus kaustophilus (GkS15), and Thermus thermophilus (TtS15) from a partially patterned RNA sequence pool. Following multiple attempts to enrich for aptamers to EcS15, I find that aptamers to this homolog are infrequent in this RNA sequence pool. I successfully enriched for Gk- and TtS15 aptamers from this sequence pool and using high-throughput sequencing and clustering analysis go on to show that these homologs have highly overlapping RNA-binding profiles, though the aptamers enriched by TtS15 exhibit slightly more sequence diversity than those enriched by GkS15. I confirm that three unique aptamers from the final RNA pools bind both homologs in vitro, and a single nucleotide change that differentiates two of these aptamers causes a decrease in affinity for TtS15 but not GkS15. This mutation causes a change in the predicted folding of these two aptamers, and greatly reduces its frequency in the population enriched by TtS15. Taken together, the work presented in this thesis shows overlapping but not identical RNA-binding profiles for the Gk- and TtS15 homologs to aptamers enriched from a partially patterned RNA library and represents the first comparative study of two homologous RNA-binding proteins using in vitro selection against an RNA library.