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Irrespective of the mode of initiation, Nsp1-mediated cleavage is induced by the cooperative action of the N-terminal domain of Nsp1, the RRM domain of eIF3g and 40S subunits. Using\n                  <jats:italic>in vitro<\/jats:italic>\n                  reconstitution, we determined that cleavage occurs by transesterification following intramolecular nucleophilic attack of the 2\u2019OH of the ribose on the adjacent phosphodiester bond yielding 5\u2019OH and 2\u2019,3\u2018-cyclic phosphate termini. Cleavage requires a guanosine \u223c10-22 nucleotides from the 5\u2019 end of mRNA, occurs within a narrow window upstream of this G, is most efficient between nucleotides at positions -6\/-7 and -7\/-8 relative to G, and shows a preference for Pu at positions -7 or -8 which provides the 2\u2019OH for the nucleophilic attack. Zero-length UV cross-linking of Nsp1 to nucleotides at positions -1 and -2 suggests that the critical guanosine may be recognized by Nsp1. Resistance to Nsp1-mediated cleavage of SARS-CoV-2 mRNA was ensured both by the relatively long distance between its G\n                  <jats:sub>23<\/jats:sub>\n                  G\n                  <jats:sub>24<\/jats:sub>\n                  and the 5\u2019end and by the preceding oligoPy stretch lacking purines at positions -7 or -8 upstream of G\n                  <jats:sub>23<\/jats:sub>\n                  G\n                  <jats:sub>24<\/jats:sub>\n                  .\n                <\/jats:p>","DOI":"10.64898\/2026.06.18.733267","type":"posted-content","created":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T04:25:21Z","timestamp":1782188721000},"source":"Crossref","is-referenced-by-count":0,"title":["Mechanism and key RNA determinants of SARS-CoV-2 Nsp1-induced endonucleolytic cleavage of mRNA"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4446-5128","authenticated-orcid":false,"given":"Irina S.","family":"Abaeva","sequence":"first","affiliation":[{"name":"Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1690-7913","authenticated-orcid":false,"given":"Atala B.","family":"Jena","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3982-2090","authenticated-orcid":false,"given":"Christopher U. 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