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SLFN14, a member of SLFN family, is associated with multiple human diseases; however, neither its functions nor its disease mechanisms are fully understood. Herein, human SLFN14 biochemically is characterized, demonstrating that it specifically cleaves RNAs containing short duplex regions, such as hairpin RNAs and tRNAs, but does not have ATPase or helicase activity. Cryogenic electron microscopy structures of SLFN14 apoenzyme (2.84 \u00c5) and SLFN14\u2010hairpin RNA complex (2.88 \u00c5) are determined, revealing that SLFN14 assembles into a ring\u2010like dimer and dimerization is mainly mediated by hydrophobic contacts. Two N\u2010terminal RNase domains of SLFN14 are organized head\u2010to\u2010tail, forming an RNA\u2010binding groove that can accommodate a 12\u2010bp hairpin RNA. The hairpin RNA is recognized mainly through phosphate backbone interactions. Further, SLFN14 is shown to inhibits HIV\u20101 pseudovirus replication. The anti\u2010HIV\u20101 activity of SLFN14 is via codon\u2010usage\u2010dependent translational inhibition and impairment of the programmed \u20101 ribosomal frameshifting, with an efficiency comparable to that of Shiftless. Using tRNA PCR arrays, SLFN14 and SLFN11 are found to decrease both nuclear\u2010encoded and mitochondrial tRNAs in cells. Together, these results provide novel insights into the function of SLFN14 and its role in HIV\u20101 restriction.<\/jats:p>","DOI":"10.1002\/advs.202501727","type":"journal-article","created":{"date-parts":[[2025,7,11]],"date-time":"2025-07-11T09:06:12Z","timestamp":1752224772000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Human Schlafen 14 Cleavage of Short Double\u2010Stranded RNAs Underpins its Antiviral Activity"],"prefix":"10.1002","volume":"12","author":[{"given":"Mengyun","family":"Li","sequence":"first","affiliation":[{"name":"NHC Key Laboratory of Systems Biology of Pathogens National Institute of Pathogen Biology Chinese Academy of Medical Sciences and Peking Union Medical College  Beijing 100730 P. R. China"},{"name":"Key Laboratory of Pathogen Infection Prevention and Control (Peking Union Medical College) Ministry of Education  Beijing 100730 P. R. 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China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6329-3582","authenticated-orcid":false,"given":"Sheng","family":"Cui","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Systems Biology of Pathogens National Institute of Pathogen Biology Chinese Academy of Medical Sciences and Peking Union Medical College  Beijing 100730 P. R. China"},{"name":"Key Laboratory of Pathogen Infection Prevention and Control (Peking Union Medical College) Ministry of Education  Beijing 100730 P. R. China"},{"name":"State Key Laboratory of Respiratory Health and Multimorbidity Chinese Academy of Medical Sciences and Peking Union Medical College  Beijing 100730 P. R. 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