UBC Theses and Dissertations
The PRMT response to inflammation : substrate methylation and alternative splicing Vhuiyan, Md. Mynol Islam
Protein arginine N-methyltransferases (PRMTs) are a family of enzymes involved in signaling pathways and gene expression by methylating arginine residues of substrate proteins. PRMT2 has been demonstrated to play a role in the NF-κB signaling pathway. Moreover, our lab recently revealed association using proteomic techniques between PRMT2 and splicing factors including Src-associated in mitosis 68 kDa protein (SAM68) that mediates the alternative splicing of BCL-X involved in the NF-κB mediated inflammatory pathway. I wanted to investigate if the PRMT activity plays a role in response to inflammation under the treatment of inflammatory cytokine tumor necrosis factor-α (TNF-α) or pro-inflammatory bacterial lipopolysaccharide (LPS) in A549 cells. My proteomic experiments revealed that TNF-α and LPS cause similar changes to arginine methylation for proteins primarily involved in mRNA processing, RNA splicing, and nuclear transport, indicating that these two inflammatory stimuli share mutual downstream pathways involving methyltransferase activity. Among the proteins that showed hypermethylation upon treatment relative to control in mass spectrometry analysis, GAP SH3 domain-binding protein 2 (G3BP2) showed consistently in all three replicates on average a 1.5-fold increase in methylation at the R468 site in both TNF-α and LPS-treated cells. G3BP2 binds to IκB-α and prevents NF-κB translocation into the nucleus for subsequent signaling. G3BP2 methylation was necessary for signaling to occur in the Wnt/β-catenin signaling pathway. Methylation of G3BP2 might also have similar role in the inflammatory pathway that demands further study. Moreover, consistent with an inflammatory response, proteins particularly involved in innate immunity and viral response increased upon TNF-α treatment that could be related to the observed change in methylation of proteins involved in RNA processing. Our finding that PRMT2 interacts with SAM68, prompted me to investigate the potential role of PRMT2 in BCL-X alternative splicing. I found that reduced expression of PRMT2 by siRNA caused a decrease in the BCL-X(L)/BCL-X(s) ratio, suggesting that PRMT2 may contribute to BCL-X alternative splicing. This effect was replicated in TNF-α or LPS stimulated cells when PRMT2 expression was reduced by shRNA, and reversed when PRMT2 expression was increased. These results indicate that PRMT2 may play a role during inflammation in alternative splicing regulation.
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