Epitranscriptomic and post-transcriptional regulation of the RNome in gram negative and firmicute bacteria

PROJECT SUMMARY / ABSTRACT Post-transcriptional regulation of gene expression has significant effects on the physiology of the bacterial cells. While much has been uncovered about post-transcriptional regulation of gene expression in bacteria, there are still some key unknowns that are hindering our ability to understand how these processes work and differ

throughout the prokaryotic domain of life. In this project, our overall goal is to expand our understanding of the diversity of these processes in gram-negative model organisms, firmicutes, and mycobacteria. Our propsosed research program has two sub-projects. Project #1 is entitled: Epitranscriptomic regulation of gene expression

throughout the prokaryotic domain. In sub-project 1, we will expand our understanding of RNA modifications within a wide range of bacterial species. RNA modifications include an isomerization or chemical additions to canonical nucleotides within RNA. Generally found in tRNA molecules, RNA modifications are critical in

promoting translational fidelity by promoting ribosome mRNA decoding errors. There are several key knowledge gaps driving our experimental approach. The regulatory role of RNA modifications, cis-acting signals that may promoting regulatory targeting of RNA modifications, and the effect of GC content on the ability of codon usage

to drive targeting of genes by modified tRNAs. We will use a multi-omics approach to address these key knowledge gaps using several bacterial model organisms: Escherichia coli, Staphylococcus aureus, Clostridioides difficile, and Mycobacterium smegmatis. Project #2 is entitled: Post-transcriptional gene regulation

via novel proteins and sRNAs in firmicutes. In sub-project 2, we will expand our understanding of the regulatory role of novel proteins and sRNAs in the regulation of the RNome in firmicutes. Much our understanding of sRNA in bacteria has come from extensive studies in model organisms. This has assisted in our understanding sRNA

regulation of bacterial physiology in general. However, recent studies have uncovered hundreds of species- specific sRNA in S. aureus. The vast majority of these are uncharacterized. Since we lack a full picture of sRNA regulons in S. aureus, our understanding of bacterial sRNA biology is incomplete. In addition, many model

organisms utilize the RNA chaperone Hfq as a sRNA co-factor in some cases to promote sRNA-mRNA-RNase interactions. However, Hfq is dispensable for growth and sRNA function. It is unknown if there are other protein factors that may act as specific-specific chaperones in S. aureus or other firmicutes. We will have preliminary

data to suggest that novel regulatory proteins may exist, in S. aureus and C. difficile, in the mRNA regulation and may also modulate levels of sRNAs. We will execute RNA-immunoprecipitation sequencing of this candidate protein in S. aureus and C. difficile to identify its RNA-interactome. We have also identified predicted sRNA

interactions with several important proteins in S. aureus and execute experiments to test this interaction.