Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.
Bottom Line: Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining.PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells.Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis.
Affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.Show MeSH
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Mentions: Lastly, we assessed whether MYCN protein may be methylated by PRMT5 using liquid chromatography – tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein from SK-N-BE(2)C cells before and after PRMT5 knockdown. This analysis gave us >53% coverage of MYCN, and identified several potential sites of arginine monomethylation and dimethylation on the MYCN protein (Figure S5). By inspecting spectra of MYCN peptides bioinformatically predicted to contain arginine methylation, we pinpointed R160, R238 and R242 as high probability sites for dimethylation. Discerning asymmetric and symmetric dimethylation was not possible from LC-MS/MS analysis. However, by manually comparing the spectra for the peptide containing R238 and R242, from control siRNA and PRMT5 siRNA treated samples, we were able to identify fragment ions with reduced mass in the PRMT5 knockdown sample, consistent with the loss of dimethylarginine at R242 (Figure 8). The double-dimethylated peptide shown in Figure 8A was not found in MYCN from PRMT5 knockdown, consistent with R242 representing a primary target for PRMT5 methyltransferase activity. However, it is important to note that our analysis was not quantitative and would therefore not allow us to identify subtler changes occurring between samples. Nevertheless, these experiments demonstrate for the first time that MYCN undergoes PTM by arginine methylation, and mechanistically implicate PRMT5 as a critical regulator of MYCN protein levels.
Affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.