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Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.

Park JH, Szemes M, Vieira GC, Melegh Z, Malik S, Heesom KJ, Von Wallwitz-Freitas L, Greenhough A, Brown KW, Zheng YG, Catchpoole D, Deery MJ, Malik K - Mol Oncol (2014)

Bottom Line: 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.By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein.Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.

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Detection of dimethylated arginine residues in MYCN. (A) LC-MS/MS MS/MS spectrum of the doubly dimethylated peptide, EPAPVPAAPASAPAAGPAVASGAGIAAPAGAPGVAPPRPGGR (m/z 1211.66, 3+) from MYCN immunoprecipitated after control siRNA knockdown. The majority of ions are due to doubly charged C-terminal y ions, which show that the two C-terminal arginine residues are dimethylated (underlined arginines are dimethylated). (B) MS/MS spectrum of the singly dimethylated peptide EPAPVPAAPASAPAAGPAVASGAGIAAPAGAPGVAPPRPGGR (m/z 1202.30, 3+) from MYCN immunoprecipitated after PRMT5 knockdown. The doubly charged C-terminal fragment ions in this spectrum are 14 m/z units (corresponding to a mass difference of 28 Da) lower than those shown in (A) as a result of the absence of a second dimethylated arginine residue, corresponding to R242 in MYCN (see Figure S5).
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fig8: Detection of dimethylated arginine residues in MYCN. (A) LC-MS/MS MS/MS spectrum of the doubly dimethylated peptide, EPAPVPAAPASAPAAGPAVASGAGIAAPAGAPGVAPPRPGGR (m/z 1211.66, 3+) from MYCN immunoprecipitated after control siRNA knockdown. The majority of ions are due to doubly charged C-terminal y ions, which show that the two C-terminal arginine residues are dimethylated (underlined arginines are dimethylated). (B) MS/MS spectrum of the singly dimethylated peptide EPAPVPAAPASAPAAGPAVASGAGIAAPAGAPGVAPPRPGGR (m/z 1202.30, 3+) from MYCN immunoprecipitated after PRMT5 knockdown. The doubly charged C-terminal fragment ions in this spectrum are 14 m/z units (corresponding to a mass difference of 28 Da) lower than those shown in (A) as a result of the absence of a second dimethylated arginine residue, corresponding to R242 in MYCN (see Figure S5).

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.


Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.

Park JH, Szemes M, Vieira GC, Melegh Z, Malik S, Heesom KJ, Von Wallwitz-Freitas L, Greenhough A, Brown KW, Zheng YG, Catchpoole D, Deery MJ, Malik K - Mol Oncol (2014)

Detection of dimethylated arginine residues in MYCN. (A) LC-MS/MS MS/MS spectrum of the doubly dimethylated peptide, EPAPVPAAPASAPAAGPAVASGAGIAAPAGAPGVAPPRPGGR (m/z 1211.66, 3+) from MYCN immunoprecipitated after control siRNA knockdown. The majority of ions are due to doubly charged C-terminal y ions, which show that the two C-terminal arginine residues are dimethylated (underlined arginines are dimethylated). (B) MS/MS spectrum of the singly dimethylated peptide EPAPVPAAPASAPAAGPAVASGAGIAAPAGAPGVAPPRPGGR (m/z 1202.30, 3+) from MYCN immunoprecipitated after PRMT5 knockdown. The doubly charged C-terminal fragment ions in this spectrum are 14 m/z units (corresponding to a mass difference of 28 Da) lower than those shown in (A) as a result of the absence of a second dimethylated arginine residue, corresponding to R242 in MYCN (see Figure S5).
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fig8: Detection of dimethylated arginine residues in MYCN. (A) LC-MS/MS MS/MS spectrum of the doubly dimethylated peptide, EPAPVPAAPASAPAAGPAVASGAGIAAPAGAPGVAPPRPGGR (m/z 1211.66, 3+) from MYCN immunoprecipitated after control siRNA knockdown. The majority of ions are due to doubly charged C-terminal y ions, which show that the two C-terminal arginine residues are dimethylated (underlined arginines are dimethylated). (B) MS/MS spectrum of the singly dimethylated peptide EPAPVPAAPASAPAAGPAVASGAGIAAPAGAPGVAPPRPGGR (m/z 1202.30, 3+) from MYCN immunoprecipitated after PRMT5 knockdown. The doubly charged C-terminal fragment ions in this spectrum are 14 m/z units (corresponding to a mass difference of 28 Da) lower than those shown in (A) as a result of the absence of a second dimethylated arginine residue, corresponding to R242 in MYCN (see Figure S5).
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.

Bottom Line: 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.By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein.Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.

Show MeSH
Related in: MedlinePlus