Structural, functional and biophysical characterization of two cryptic RNA methyltransferases (Rv3366 and Rv3919c) of...

RNA methyltransferases (MTases) play a significant role in post-transcriptional regulation in bacteria, significantly influencing virulence, stress adaptation and host-pathogen interactions. In Mycobacterium tuberculosis, several putative MTases remain functionally uncharacterized. This study aims to elucidate the structural, biophysical and functional properties of two predicted M. tuberculosis RNA MTases, Rv3366 and Rv3919c, to better understand their roles in mycobacterial physiology and pathogenicity. The genes encoding Rv3366 and Rv3919c were cloned, heterologously expressed in Escherichia coli and purified using affinity chromatography. Enzymatic activity was evaluated using a methyltransferase-Glo^™ assay with S-adenosylmethionine (SAM) as the methyl donor. Biophysical and structural characterization was carried out using circular dichroism (CD) spectroscopy, fluorescence spectroscopy and thermal shift assays. Both proteins exhibited SAM-dependent MTase activity along with appreciable RNA-binding affinity. CD spectral analysis revealed a predominantly α-helical secondary structure, while fluorescence and thermal shift assays confirmed proper folding and notable thermal stability. Collectively, Rv3366 and Rv3919c display hallmark characteristics of functional RNA MTases, suggesting their involvement in RNA modification pathways that may contribute to M. tuberculosis virulence and adaptation. These findings establish a framework for future studies to explore their mechanistic roles and evaluate their potential as therapeutic targets.