Mycobacterium tuberculosis virulence-associated small RNA MTS1338 is posttranscriptionally regulated by the ribonuclease YbeY.

The bacterial protein YbeY is essential for ribosome biogenesis, heat stress response, small RNA regulation, and virulence, with its deletion proving lethal or severely detrimental to cell viability. Despite its importance, the role of YbeY in Mycobacterium tuberculosis remains unclear. In this study, we purified mycobacterial YbeY (MtbYbeY), characterized its catalytic properties, and investigated its role in regulating the virulence-associated small RNA MTS1338. MtbYbeY exhibits in vitro rRNA degradation and metal-dependent phosphodiesterase activity. It degrades MTS1338 into distinct RNA fragments, and its overexpression leads to a marked reduction in MTS1338 levels in vivo. This degradation is further supported by their inverse abundance under acidic conditions. Our findings reveal a previously unrecognized ribonuclease-mediated mechanism of small RNA regulation in M. tuberculosis. Impact statement MTS1338 is essential for mycobacterial latency and virulence. While response regulators promote its expression, this study reveals that YbeY, a ribonuclease, negatively regulates MTS1338 post-transcriptionally. Depletion of YbeY leads to intracellular accumulation of MTS1338 during host infection, highlighting a novel layer of small RNA regulation in M. tuberculosis.