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Physiologically, APOBEC3A functions in innate immunity and aberrant deamination is associated with cytosine mutations in enzymatically preferred YTCW substrate motifs in multiple cancers. Much less is known about the potential contribution of APOBEC3A-catalyzed RNA editing to virus and cancer evolution. Here, we present HAMMER (hairpin-based APOBEC3A-mediated messenger RNA editing reporter), a rapid luminescence-based cellular assay for measuring RNA editing by APOBEC3A. HAMMER reports APOBEC3A activity as a reduction in the ratio of firefly to renilla luciferase activity. Briefly, tandem renilla and firefly luciferase open reading frames are separated by an optimal APOBEC3A hairpin substrate, in which C-to-U editing of a\u00a0CGA motif yields a UGA stop codon thus preventing translation of the downstream firefly luciferase reporter, without impacting the upstream renilla reporter. HAMMER activation is dose-responsive, catalytic activity-dependent, and specific to human APOBEC3A. A panel of herpesviral ribonucleotide reductase constructs was used to show that direct inhibition of APOBEC3A results in a dose-responsive recovery of firefly luciferase expression. HAMMER is therefore a scalable and easy-to-use method for quantifying cellular APOBEC3A RNA editing activity and characterizing inhibitors.<\/jats:p>","DOI":"10.1093\/nar\/gkag302","type":"journal-article","created":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T12:42:02Z","timestamp":1774010522000},"source":"Crossref","is-referenced-by-count":5,"title":["HAMMER: hairpin-based APOBEC3A-mediated mRNA editing reporter"],"prefix":"10.1093","volume":"54","author":[{"given":"Yanjun","family":"Chen","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Structural Biology, University of Texas San Antonio , San Antonio, TX 78229 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher D","family":"Mullally","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Structural Biology, University of Texas San Antonio , San Antonio, TX 78229 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bojana","family":"Stefanovska","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Structural Biology, University of Texas San Antonio , San Antonio, TX 78229 ,","place":["United States"]},{"name":"Howard Hughes Medical Institute, University of Texas San Antonio , San Antonio, TX 78229 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9034-9112","authenticated-orcid":false,"given":"Reuben S","family":"Harris","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Structural Biology, University of Texas San Antonio , San Antonio, TX 78229 ,","place":["United States"]},{"name":"Howard Hughes Medical Institute, University of Texas San Antonio , San Antonio, TX 78229 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,4,2]]},"reference":[{"key":"2026040207444816000_B1","doi-asserted-by":"publisher","first-page":"131","DOI":"10.1016\/j.virol.2015.03.012","article-title":"APOBECs and virus restriction","volume":"479\u201380","author":"Harris","year":"2015","journal-title":"Virology"},{"key":"2026040207444816000_B2","doi-asserted-by":"publisher","first-page":"100909","DOI":"10.1016\/j.jbc.2021.100909","article-title":"Structural basis of substrate specificity in human cytidine deaminase family APOBEC3s","volume":"297","author":"Hou","year":"2021","journal-title":"J Biol Chem"},{"key":"2026040207444816000_B3","doi-asserted-by":"publisher","first-page":"390","DOI":"10.3390\/v13030390","article-title":"APOBECs and herpesviruses","volume":"13","author":"Cheng","year":"2021","journal-title":"Viruses"},{"key":"2026040207444816000_B4","doi-asserted-by":"publisher","first-page":"12688","DOI":"10.1021\/acs.chemrev.6b00296","article-title":"Functions and malfunctions of mammalian DNA-cytosine deaminases","volume":"116","author":"Siriwardena","year":"2016","journal-title":"Chem. 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