@article{0a6c7d7c3cc74455ae935189194bc004,
title = "sncRNA-1 Is a Small Noncoding RNA Produced by Mycobacterium tuberculosis in Infected Cells That Positively Regulates Genes Coupled to Oleic Acid Biosynthesis",
abstract = "Nearly one third of the world{\textquoteright}s population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different Mtb encoded proteins in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. We performed RNA sequencing and identified a subset of Mtb H37Rv-encoded small RNAs (<30 nts in length) that were produced in infected macrophages. Designated as smaller noncoding RNAs (sncRNAs), three of these predominated the read counts. Each of the three, sncRNA-1, sncRNA-6, and sncRNA-8 had surrounding sequences with predicted stable secondary RNA stem loops. Site-directed mutagenesis of the precursor sequences suggest the existence of a hairpin loop dependent RNA processing mechanism. A functional assessment of sncRNA-1 suggested that it positively regulated two mycobacterial transcripts involved in oleic acid biosynthesis. Complementary loss- and gain- of-function approaches revealed that sncRNA-1 positively supports Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Overall, the findings reveal that Mtb produces sncRNAs in infected cells, with sncRNA-1 modulating mycobacterial gene expression including genes coupled to oleic acid biogenesis.",
keywords = "Mycobacterium tuberculosis, gene regulation, miRNAs, oleic acid, small RNAs",
author = "Coskun, {Fatma S.} and Shashikant Srivastava and Prithvi Raj and Igor Dozmorov and Serkan Belkaya and Smriti Mehra and Golden, {Nadia A.} and Bucsan, {Allison N.} and Chapagain, {Moti L.} and Wakeland, {Edward K.} and Deepak Kaushal and Tawanda Gumbo and {van Oers}, {Nicolai S.C.}",
note = "Funding Information: We have focused our functional studies sncRNA-1 since it was not identified in any prior transcriptome studies and it exists within a key pathogenicity locus, RD1. RNA-seq of the fast-growing auxotroph mutant of Mtb H37Rv 6230 over-expressing sncRNA-1 suggested a novel role for this sncRNA in the positive regulation of two genes involved in oleic acid biogenesis, Rv1094 and Rv0242c. This was confirmed for Rv0242c, a gene that encodes FabG4, a non-canonical and essential 3-oxoacyl-thioester reductase (Gurvitz, 2009). Rv0242c has been implicated in mycobacterial resistance to streptomycin (Sharma et al., 2010). Regulation of Rv0242c by sncRNA-1 indicates a possible role for this sRNA in mycobacterial drug resistance. The production of oleic acid, an unsaturated fatty acid, also contributes to decreased membrane fluidity in the mycobacteria, improving their survival chances in harsh conditions such as the phagolysosome in macrophages (Bloch and Segal, 1956; Lee et al., 2013). Our findings suggest that sncRNA-1 regulates Rv0242c expression by direct sequence specific interactions involving a putative seed sequence in sncRNA-1, which is conserved among mycobacteria. This was supported by the mutagenesis of the 5′ UTR region of Rv0242c. The 5′ UTR region has a critical role in gene expression, which encompasses the RBS (Flentie et al., 2016). RNAfold was used to compare the predicted secondary structure of this region for the wild type Rv0242c along with the two mutants that we created (Figure 5A). A bending of the RBS was revealed that was unique to Rv0242c_M2, implying that the access of the translational machinery may be blocked, hence reducing Rv0242c_M2 stability and expression (Figure 5A and Supplementary Figure S5B). Overall, these data suggest that sncRNA-1 regulates the expression of Rv0242c through its 5′ UTR. Future studies will address the mechanism of regulation of Rv1094. Funding Information: We would like to thank Dr. E. von Grote, Ms. J. Eitson, and Ms. J. MacLeod for their excellent technical work. We appreciate the intellectual input from Drs. A. Hoover, Q. Du, M. de la Morena, S. Khan, E. Hansen Sebastien Winter, and Nicholas Conrad (UT Southwestern Medical Center). We thank Drs. Eric Olson and Lora Hooper (UT Southwestern Medical Center) for reviewing the manuscript, and Dr. Kristen Arnvig (University College London) for graciously providing the pKA-303 vector. Drs. Torin Weisbrod and William Jacobs (Albert Einstein College of Medicine) were very generous in providing the auxotroph mutants of Mtb H37Rv. We appreciate the helpful discussions from Dr. C. Eiken (LC Sciences, Houston, TX, United States). Ken Taylor (Exiqon Inc., Woburn, MA, United States, now part of Qiagen) provided technical information and support for developing the locked nucleic acid inhibitors. Funding. This work was supported, in part by High Risk/High Impact and Beecherl grants from the University of Texas Southwestern Medical Center to NvO; a National Institutes of Health/National Institute of General Medical Sciences Director New Innovator Award (1 DP2 OD001886) and an intramural grant from the Baylor Research Institute (BRI) to TG while he was a member of the BRI; and an NIH, National Institute of Allergy and Infectious Disease grant to DK (AI089323). Publisher Copyright: {\textcopyright} Copyright {\textcopyright} 2020 Coskun, Srivastava, Raj, Dozmorov, Belkaya, Mehra, Golden, Bucsan, Chapagain, Wakeland, Kaushal, Gumbo and van Oers.",
year = "2020",
month = jul,
day = "28",
doi = "10.3389/fmicb.2020.01631",
language = "English (US)",
volume = "11",
journal = "Frontiers in Microbiology",
issn = "1664-302X",
publisher = "Frontiers Media S. A.",
}