Identification of Smaller Noncoding RNAs Produced by Mycobacterium Tuberculosis in Infected Macrophages That Regulate Mtb Growth and Survival

It is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different proteins encoded by Mtb in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. A subset includes small RNAs (sRNAs) between the sizes of 50-350 nts. The current study focused on the identification and characterization of miRNA-like sRNAs <50 nts produced by Mtb. A sRNA-centered RNA-sequencing approach was performed and a subset of Mtb-encoded smaller noncoding RNAs (sncRNAs) were identified. Thirty-five distinct Mtb-encoded sncRNAs were discovered, with most being induced in infected eukaryotic cells. Three sncRNAs, sncRNA-1, sncRNA-6, and sncRNA-8, predominated the read counts. They were contained in longer RNA transcripts with stable secondary RNA stem loops and structures like precursor microRNAs. My work established that sncRNA-1 positively regulates two mycobacterial transcripts involved in oleic acid biosynthesis. Loss- and gain- of-function approaches reveal that sncRNA-1 enhances Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Given evidence that RNA processing enzymes were involved in the formation of the sncRNAs, different components of core RNA degradosome were characterized for their ability to process the precursor forms of the sncRNAs. My work revealed that PNPase degrades sncRNA-8 and preliminary evidence suggests that sncRNA-1 is also likely a target, which could be critical in the oleic acid deficient media. Overall, my study reveals that Mtb produces a set of sncRNAs in infected cells, with one modulating mycobacterial gene expression and mycobacterial pathogenicity coupled to oleic acid biogenesis. Future studies will address the functions of other sncRNAs and focus on the identification of sncRNA processing enzymes.