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Dhh1 has been shown to interact with the MIF4G domain of the central scaffold protein of the Ccr4\u2013Not deadenylase complex, Not1, as well as the decapping activator Edc3. Although structures have been published of Dhh1 in complex with Not1MIF4G or an Edc3 peptide, the impact of these interactions on the catalytic cycle of Dhh1 are unknown. Here, we show that cEdc3 enhances ATP and RNA binding by Dhh1, whereas Not1MIF4G promotes the catalytic step of ATP hydrolysis. Additionally, the modulation of Dhh1 activity by Edc3 requires a more extensive set of interaction motifs and interfaces than was previously recognized. While the effect of either Not1MIF4G or Edc3 on the ATPase activity of Dhh1 is modest, together both proteins increase Dhh1 activity over 200-fold. The fact that Dhh1 biochemical activity is cooperatively tuned by deadenylation and decapping factors suggests that Dhh1 may coordinate deadenylation and decapping in the 5\u2032-3\u2032 mRNA decay pathway through changes in its ATP-coupled RNA binding affinity during its catalytic cycle.<\/jats:p>","DOI":"10.1093\/nar\/gkag908","type":"journal-article","created":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T13:53:33Z","timestamp":1788616413000},"source":"Crossref","is-referenced-by-count":0,"title":["Deadenylation and decapping factors cooperatively stimulate biochemical activities of DEAD-box ATPase Dhh1"],"prefix":"10.1093","volume":"54","author":[{"given":"Gabriel A","family":"Braun","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Chemistry, University of California , San Francisco, CA 94158 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rakesh","family":"Kumar","sequence":"additional","affiliation":[{"name":"Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health , Bethesda, MD 20892 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1627-8395","authenticated-orcid":false,"given":"Alan G","family":"Hinnebusch","sequence":"additional","affiliation":[{"name":"Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health , Bethesda, MD 20892 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1377-4705","authenticated-orcid":false,"given":"John 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