Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Breaker, Ronald

Abstract

Bacteria are under significant evolutionary pressures to maintain compact genomes.Therefore, they typically have high coding density compared to eukaryotes. As a result, large and highly structured noncoding RNAs (ncRNAs) are extremely rare in bacteria. However, the few classes of known large ncRNAs have unique and fundamental biological roles and are often catalytic. Furthermore, these ncRNAs reveal what may have been possible under a hypothetical ancient RNA world in which all major biological functions in existing organisms were performed by RNA, rather than DNA and protein. Further study of large ncRNAs is likely to reveal new insights into RNA catalysis and the biology of both modern and ancient organisms. OLE (ornate, large, extremophilic) RNAs are among the best studied bacterial large ncRNAs whose function(s) are yet unknown. Bacteria across the superphylum Bacil- lota are known to have OLE RNA. Experiments in the OLE-containing haloalkaliphile Halalkalibacterium halodurans have shown that OLE RNA exists in a ribonucleoprotein (RNP) complex with at least three proteins, called OapA, OapB, and OapC, where ”Oap” stands for OLE-associated protein. Furthermore, it is known that genetic disruption of components of the OLE RNP complex does not produce noticeable phenotypes in stan- dard growth conditions, but leads to growth defects under several environmental stress conditions including Mg2+ stress (>4 mM), cold stress (~20°C), ethanol stress (5%), and exposure to alternate carbon sources. However, the biological and biochemical functions of OLE RNA are still unclear. In Chapter 2, I describe my efforts to gain insights into OLE RNA biology using bioin- formatics. I used a technique called phylogenetic profiling to identify protein-coding genes that either correlate or anticorrelate with the presence of OLE RNA across Bacillota. These efforts demonstrated that OLE RNA correlates with all three known OLE-associated pro- teins, suggesting that there is evolutionary pressure for OLE-containing organisms to also maintain these associated proteins. Additionally, I show that OLE RNA anticorrelates with a Mg2+ transporter called MpfA, which is also a distant homolog of OapA. These results suggest the OLE RNP complex may be functionally complementary with MpfA. Indeed, in Chapter 3, I show that genetic disruption of either MpfA or the OLE RNP complex leads to a Mg2+ stress phenotype that can be rescued by the other. In Chapter 4, I show work exploring connections between Mg2+ stress and central car- bon metabolism using transcriptomics. I show that the Mg2+ excess growth defect can be rescued by Mn2+ supplementation. Furthermore, I show that Mg2+, cold, and ethanol stress all lead to differential regulation of similar categories of genes in ∆ole cells, sug- gesting perhaps that these major stress conditions may have similar or at least overlapping mechanisms. In Chapter 5, I demonstrate work describing the interactions between OLE RNA and BH3508, which is a restriction modification system cytosine methyltransferase and puta- tive interacting partner of OLE RNA. I show that BH3508 binds OLE RNA in vitro with high affinity but low specificity, suggesting that BH3508 is unlikely to be a natural partner of the OLE RNP complex. Finally, in Chapter 6, I review major current gaps in our understanding of OLE RNA biology and novel strategies that can be used to answer outstanding questions.

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