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Updated: Apr 11, 2026
Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling
Published on: October 28, 2014
5-Azacytidine incorporation into mRNAs disrupts translation and induces ribosome collisions
Alexis B Roberson1, James Marks1, Ruby Pitts1
1Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232.
Abstract:
5-Azacytidine (5-AzaC) is a cytidine analog and is widely used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). 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's mechanism of action, alongside its known DNA-mediated effects.
Insights
5-Azacytidine (5-AzaC) disrupts protein synthesis by incorporating into RNA, causing translation errors and activating stress responses, contributing to its cell-killing effects beyond DNA modification.
Area of Science:
- Molecular Biology
- Cancer Therapeutics
- Epigenetics
Background:
- 5-Azacytidine (5-AzaC) is a key drug for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML).
- While DNA incorporation causes hypomethylation, most 5-AzaC incorporates into RNA, with unknown functional impacts.
- Understanding RNA-mediated effects is crucial for optimizing 5-AzaC therapy.
Purpose of the Study:
- To investigate the functional consequences of 5-AzaC incorporation into RNA.
- To elucidate the impact of RNA-incorporated 5-AzaC on protein synthesis and cellular signaling.
- To explore the role of translation disruption in 5-AzaC's cytotoxicity.
Main Methods:
- Utilized Ribosome profiling sequencing (Ribo-seq), Disome-seq, and RNA-seq.
- Analyzed mRNA modifications and ribosome occupancy.
- Investigated stress response activation (ISR, RSR) and Ribosome Quality Control (RQC) pathways.
Main Results:
- 5-AzaC rapidly incorporates into mRNA, causing C-to-G transversions and inhibiting protein synthesis within 2 hours.
- Disome footprints accumulate at specific C-rich codons, indicating translation stalling and disruption.
- 5-AzaC activates integrated stress response (ISR) and ribotoxic stress response (RSR) in a GCN2- and ZAK-dependent manner.
- Loss of ZNF598 sensitizes cells to 5-AzaC, highlighting the role of Ribosome Quality Control.
Conclusions:
- 5-AzaC incorporation into RNA disrupts mRNA decoding and triggers disome-mediated signaling.
- Translation disruption is a significant mechanism contributing to 5-AzaC's cytotoxicity, complementing its DNA-based effects.
- These findings offer a new perspective on 5-AzaC's action and potential therapeutic strategies.
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