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Although its therapeutic activity is primarily attributed to hypomethylation resulting from DNA incorporation, the majority of 5-AzaC is incorporated into RNA. However, the functional consequences of 5-AzaC incorporation into RNA have been unknown. Here, we show that 5-AzaC treatment of cells leads to inhibition of protein synthesis. Ribo-seq, Disome-seq, and RNA-seq in cells treated with 5-AzaC exhibit a time-dependent C-to-G transversion signature in mRNAs within 2 h of treatment. These transversion events are enriched within footprint positions corresponding to the A-site of monosomes or leading stalled ribosome in a disome complex. Consistently, ribosome and disome footprints are accumulated at sites with C-rich codons in the A-site, specifically with the codons containing a C in the second position. 5-AzaC activates the integrated stress response (ISR) and the ribotoxic stress response (RSR) in a GCN2- and ZAK-dependent manner, consistent with disome-mediated signaling. Furthermore, loss of the Ribosome Quality Control (RQC) factor, ZNF598, sensitizes cells to 5-AzaC. Collectively, our results support a model where 5-AzaC is rapidly incorporated into mRNAs, disrupts decoding, and triggers disome-mediated signaling pathways, which contribute to its cytotoxicity. These findings suggest that translation disruption represents an additional layer of 5-AzaC\u2019s mechanism of action, alongside its known DNA-mediated effects.<\/jats:p>","DOI":"10.64898\/2026.03.30.714548","type":"posted-content","created":{"date-parts":[[2026,4,1]],"date-time":"2026-04-01T04:00:42Z","timestamp":1775016042000},"source":"Crossref","is-referenced-by-count":1,"title":["5-Azacytidine incorporation into mRNAs disrupts translation and induces ribosome collisions"],"prefix":"10.64898","author":[{"given":"Alexis B","family":"Roberson","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James","family":"Marks","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruby","family":"Pitts","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bavavarshini","family":"Tamilselvam","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brian","family":"Grieb","sequence":"additional","affiliation":[{"name":"Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William P","family":"Tansey","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232"},{"name":"Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, 37232"},{"name":"Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, 37240"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2504-5489","authenticated-orcid":false,"given":"Sezen","family":"Meydan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232"},{"name":"Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, 37235"},{"name":"Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, 37232"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026040311150659000_2026.03.30.714548v1.1","doi-asserted-by":"publisher","DOI":"10.1007\/s00018-016-2261-7"},{"key":"2026040311150659000_2026.03.30.714548v1.2","doi-asserted-by":"publisher","DOI":"10.1080\/10409230802594043"},{"key":"2026040311150659000_2026.03.30.714548v1.3","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.REV119.006513"},{"key":"2026040311150659000_2026.03.30.714548v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/J.CELL.2025.01.005"},{"key":"2026040311150659000_2026.03.30.714548v1.5","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1146\/annurev-cellbio-111822-113326","article-title":"Ribosome Assembly and Repair","volume":"40","year":"2024","journal-title":"Annu Rev Cell Dev Biol"},{"key":"2026040311150659000_2026.03.30.714548v1.6","doi-asserted-by":"crossref","unstructured":"Huang, H.Y. , and Hopper, A.K . 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