VapCs of Mycobacterium tuberculosis Cleave RNAs Essential for Translation

The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin-Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in Enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by site-specific cleavage of the universally conserved Sarcin-Ricin loop (SRL) in 23S rRNA. Here we identify cleavage targets for 12 VapCs from M. tuberculosis by applying UV-crosslinking and deep sequencing. Remarkably, these VapCs are all endoribo-nucleases that cleave RNA targets that are essential for decoding at the ribosomal A-site. Eleven VapCs cleave specific tRNAs while one exhibits SRL cleavage activity. These findings suggest that multiple vapBC modules contribute to the survival of M. tuberculosis in its human host by reducing the level of translation. Overall design: Six VapC toxins were tagged with the dual affinity tag His-TEV-FLAG tag and expresed in Mycobacterium smegmatis. VapC-HTF expression was induced at OD600 0.3-0.4 for 20min and irradiated with 1800mJ of UV-C for 100seconds. Cells harvested and washed with ice cold PBS before snap freezing in liquid nitrogen. RNA-VapC complexes wer purified over M2-anti-FLAG resin and Ni-NTA resin. Sequencing libraries were prepared from purified complexes essentailly as per Tree et al. Mol Cell 2014. Amplified libraries were sequenced on an Illumina MiSeq instrument.