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These TA systems have been implicated in establishing the nonreplicating persistent state of this pathogen during latent tuberculosis infection. More than half of the <jats:italic>M. tuberculosis<\/jats:italic> TA systems belong to the VapBC (<jats:underline>v<\/jats:underline>irulence <jats:underline>a<\/jats:underline>ssociated <jats:underline>p<\/jats:underline>rotein) family. In this work, we first identified the RNA targets for the <jats:italic>M. tuberculosis<\/jats:italic> VapC-mt11 (VapC11, Rv1561) toxin <jats:italic>in vitro<\/jats:italic> to learn more about the general function of this family of toxins. Recombinant VapC-mt11 cleaved 15 of the 45\u2009<jats:italic>M. tuberculosis<\/jats:italic> tRNAs at a single site within their anticodon stem loop (ASL) to generate tRNA halves. Cleavage was dependent on the presence of a GG consensus sequence immediately before the cut site and a structurally intact ASL. However, in striking contrast to the broad enzyme activity exhibited <jats:italic>in vitro<\/jats:italic>, we used a specialized RNA-seq method to demonstrate that tRNA cleavage was highly specific <jats:italic>in vivo<\/jats:italic>. Expression of VapC-mt11 in <jats:italic>M. tuberculosis<\/jats:italic> resulted in cleavage of only two tRNA isoacceptors containing the GG consensus sequence, tRNA<jats:sup>Gln32-CUG<\/jats:sup> and tRNA<jats:sup>Leu3-CAG<\/jats:sup>. Therefore, our results indicate that although <jats:italic>in vitro<\/jats:italic> studies are useful for identification of the class of RNA cleaved and consensus sequences required for accurate substrate recognition by endoribonuclease toxins, definitive RNA target identification requires toxin expression in their native host. The restricted <jats:italic>in vivo<\/jats:italic> specificity of VapC-mt11 suggests that it may be enlisted to surgically manipulate pathogen physiology in response to stress.<\/jats:p>","DOI":"10.1038\/s41598-019-41548-9","type":"journal-article","created":{"date-parts":[[2019,4,11]],"date-time":"2019-04-11T10:15:44Z","timestamp":1554977744000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":36,"title":["Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host"],"prefix":"10.1038","volume":"9","author":[{"given":"Melvil\u00ed","family":"Cintr\u00f3n","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ju-Mei","family":"Zeng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7410-1598","authenticated-orcid":false,"given":"Valdir C.","family":"Barth","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan W.","family":"Cruz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert N.","family":"Husson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nancy A.","family":"Woychik","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2019,4,11]]},"reference":[{"key":"41548_CR1","doi-asserted-by":"publisher","first-page":"33","DOI":"10.1093\/protein\/gzq081","volume":"24","author":"VL Arcus","year":"2011","unstructured":"Arcus, V. 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