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PRMT5 dimethylates R30 of the p65 subunit to activate NF-κB
Han Wei1, Benlian Wang, Masaru Miyagi
1Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Abstract:
The ubiquitous inducible transcription factor NF-κB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-κB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-κB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1β, the p65 subunit of NF-κB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-κB to bind to κB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-κB activity, whereas PRMT5 knockdown had the opposite effect. Microarray analysis revealed that ∼85% of the NF-κB-inducible genes that are down-regulated by the R30A mutation are similarly down-regulated by knocking PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-κB-inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-κB-induced gene expression.
Insights
Protein-arginine methyltransferase 5 (PRMT5) dimethylates NF-κB p65 at arginine 30, enhancing immune responses and gene expression. This modification is crucial for NF-κB DNA binding and is implicated in cancer progression due to PRMT5 overexpression.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Nuclear factor-kappa B (NF-κB) is a key regulator of immune and inflammatory responses, as well as tumorigenesis.
- Posttranslational modifications, including phosphorylation, acetylation, ubiquitination, and lysine methylation, extensively control NF-κB activity.
- Arginine methylation of NF-κB has not been previously characterized.
Purpose of the Study:
- To investigate the role of arginine methylation in the regulation of NF-κB activity.
- To identify the specific arginine residues and methyltransferases involved in NF-κB modification.
- To elucidate the functional consequences of arginine methylation on NF-κB DNA binding and gene expression.
Main Methods:
- Utilized IL-1β stimulation to induce NF-κB activity.
- Employed site-directed mutagenesis to create arginine-to-alanine (R30A) and arginine-to-lysine (R30K) p65 mutants.
- Performed co-immunoprecipitation assays to identify interacting arginine methyltransferases.
- Conducted microarray analysis to assess changes in NF-κB-inducible gene expression.
- Utilized computational modeling to predict the structural impact of arginine methylation.
Main Results:
- Identified arginine 30 (R30) on the p65 subunit of NF-κB as a site of dimethylation.
- Demonstrated that protein-arginine methyltransferase 5 (PRMT5) is responsible for the dimethylation of R30.
- Showed that R30A and R30K mutations significantly impair NF-κB DNA binding and transcriptional activity.
- Overexpression of PRMT5 enhances NF-κB activity, while PRMT5 knockdown reduces it.
- Found that approximately 85% of NF-κB-inducible genes downregulated by the R30A mutation are also downregulated by PRMT5 knockdown.
- Computational modeling suggests dimethyl R30 stabilizes p65 structure, enhancing DNA affinity.
Conclusions:
- Arginine methylation of NF-κB p65 at R30 by PRMT5 is a novel regulatory mechanism.
- This modification is critical for optimal NF-κB DNA binding and the induction of target genes, including cytokines and chemokines.
- Aberrant PRMT5 overexpression in cancer may contribute to tumorigenesis by enhancing NF-κB-driven gene expression.
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