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Updated: Dec 25, 2025
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells
Cristina Santoriello1,2, Audrey Sporrij1,2, Song Yang1,2
1Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
Abstract:
The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.
Insights
Leflunomide disrupts neural crest development by reducing pyrimidines. Progesterone signaling and the RNA helicase DDX21 were found to counteract this effect, revealing DDX21 as a key mediator of transcription during nucleotide stress.
Area of Science:
- Molecular Biology
- Developmental Biology
- Biochemistry
Background:
- Nucleotide availability critically influences transcription processes.
- Inhibition of dihydroorotate dehydrogenase (DHODH) by leflunomide depletes pyrimidine pools, impacting gene transcription.
- Leflunomide treatment impairs neural crest development and melanoma growth by blocking transcription elongation.
Purpose of the Study:
- To elucidate the mechanism of action of leflunomide on neural crest development.
- To identify factors that can rescue leflunomide-induced transcriptional defects.
- To understand the role of nucleotide availability in regulating transcription and cellular stress responses.
Main Methods:
- In vivo chemical suppressor screen in zebrafish to identify genetic modifiers of leflunomide's effects.
- Analysis of progesterone and progesterone receptor (Pgr) signaling pathways.
- Proteomic analysis to identify protein interactions.
- Assessment of DDX21 (DEAD-box helicase 21) function in zebrafish and human melanoma cells.
- Chromatin occupancy studies and gene expression analysis.
Main Results:
- Alterations in progesterone/Pgr signaling suppressed leflunomide-induced neural crest defects in zebrafish.
- Progesterone rescued transcriptional elongation defects in zebrafish models of Paf complex deficiency.
- Proteomics identified a direct interaction between Pgr and the RNA helicase DDX21.
- DDX21-deficient zebrafish exhibited resistance to leflunomide-induced stress.
- Nucleotide depletion reduced DDX21 chromatin occupancy, while supplementation reversed leflunomide-induced changes.
Conclusions:
- DDX21 functions as a critical sensor and mediator of transcription in response to nucleotide stress.
- Progesterone signaling and DDX21 play protective roles against leflunomide-induced transcriptional dysregulation.
- These findings reveal a novel mechanism linking nucleotide metabolism, transcription, and developmental processes.
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