| Summary: | Pseudouridine (Ψ) is an ubiquitous RNA modification, present in the tRNAs and rRNAs of species across all domains of life, and is now known to be present in the mRNAs of diverse eukaryotes. However, the modification was yet to be identified in bacterial mRNAs, and its functional roles remain largely unknown.
In Chapter 1, I provide an overview of the structure and function of pseudouridine, focusing on the properties that can impact the structure, translation, and interactome of the mRNAs that contain it. I give a brief review of the enzymes that carry out tRNA and rRNA modification, which are likely to also modify mRNAs. I also summarize current knowledge about mRNA modifications in bacteria.
In Chapter 2, I report the discovery of pseudouridines in E. coli mRNA, located in coding sequences as well as 5′ and 3′ untranslated regions, and estimate that there are between 100 and 150 pseudouridine sites in mRNA under the growth conditions profiled. By testing the mRNA modification capacity of all 11 pseudouridine synthases, I identify RluA as the predominant mRNA-modifying enzyme, modifying the majority of high-confidence sites. Additionally, the enzymes RluC and RluD also carry out reproducible modification of a few mRNAs. Using RNA structure probing data to inform secondary structure prediction, I show that all mRNA targets of RluA share a common sequence and structural motif, which also occurs in its canonical tRNA and rRNA targets. A significant mRNA target of RluA is the 5′ UTR of a transcript encoding the components of the type 1 pilus. Knocking out RluA led to upregulation of these genes, and an increase in cell motility.
In Chapter 3, I discuss the possible functional consequences of mRNA pseudouridylation, and discuss the potential roles of Ψ in pilus expression in greater depth. Additionally, since I discovered sparse but reproducible modification by RluC and RluD, I compare the known structure and specificity of these three mRNA:Ψ synthases, and discuss the potential basis for their different pseudouridylation landscapes in mRNA.
Overall, this work identifies pseudouridine in mRNAs encoding critical proteins and demonstrates the capacity of Ψ to regulate the transcripts that contain it. In doing so, it expands the known bacterial epitranscriptome and provides insight into the ways RNA modifications are leveraged for gene regulation.
|
|---|