biostudies-literatureFrazier MNNational Institute of Environmental Health SciencesNational Institute of Allergy and Infectious DiseasesIntramural NIH HHSUS National Institutes of Health Intramural Research Program10136-10149https://www.ebi.ac.uk/biostudies/studies/S-EPMC8385992biostudies-literatureUnknown49(17)Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3' of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here, we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15Nucleic acids researchCharacterization of SARS2 Nsp15 nuclease activity reveals it's mad about U.PMC8385992ZIA ES1032471ZIAES1033401ZI CES1032061ZI CES102488Z01 ES043010ZIA ES103340ZIC ES102488ZIC ES103326ZIC ES103206Deterding LJWilliams JGStanley REKrahn JMPerera LBorgnia MJDillard LBWilson IMPillon MCFrazier MNStewart ZDfalseCharacterization of SARS2 Nsp15 nuclease activity reveals it's mad about U.Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3' of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here, we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp152021-01-01T00:00:00Z2021 Sep2026-04-08T08:15:20.274Z2022-02-11T15:01:02.517ZS-EPMC83859923440346610.1093/nar/gkab719