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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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MYCN depletion triggered by PRMT5 knockdown. (A) Immunoblot analysis showing MYCN depletion in SK-N-BE(2)C, NGP and Kelly NB cell-lines following transfection with two independent PRMT5 siRNAs, negating the possibility of off-target effects. (B) PRMT5 knockdown in the SHEP-Tet21N cell-line harbouring inducible MYCN expression. MYCN is switched off in the presence of tetracycline (+Tet) and induced after its removal (−Tet). Cell death is clearly visible in the MYCN-on cells after PRMT5 knockdown. (C) Cell counts showing significantly increased cell death (asterisked, P < 0.005, Student's t-test) after PRMT5 knockdown in cells induced to express MYCN. A statistically insignificant change (not significant, ns) is observed in MYCN-off cells. (D) Immunoblots of SHEP-Tet21N cells demonstrating that PRMT5 also affects MYCN expressed from the inducible transgene. Depletion is accompanied by an increase in cleaved PARP indicative of apoptosis.
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fig4: MYCN depletion triggered by PRMT5 knockdown. (A) Immunoblot analysis showing MYCN depletion in SK-N-BE(2)C, NGP and Kelly NB cell-lines following transfection with two independent PRMT5 siRNAs, negating the possibility of off-target effects. (B) PRMT5 knockdown in the SHEP-Tet21N cell-line harbouring inducible MYCN expression. MYCN is switched off in the presence of tetracycline (+Tet) and induced after its removal (−Tet). Cell death is clearly visible in the MYCN-on cells after PRMT5 knockdown. (C) Cell counts showing significantly increased cell death (asterisked, P < 0.005, Student's t-test) after PRMT5 knockdown in cells induced to express MYCN. A statistically insignificant change (not significant, ns) is observed in MYCN-off cells. (D) Immunoblots of SHEP-Tet21N cells demonstrating that PRMT5 also affects MYCN expressed from the inducible transgene. Depletion is accompanied by an increase in cleaved PARP indicative of apoptosis.

Mentions: To further investigate the strong association between PRMT5 and MYCN status, we next examined the effect of PRMT5 knockdown on MYCN in SK-N-BE(2)C, NGP and Kelly cell-lines, all of which have MYCN amplification. As shown in Figure 4A, PRMT5 depletion was accompanied by a dramatic decrease of MYCN protein with two independent PRMT5 siRNAs, thereby negating the possibility of off-target effects. Notably, the PRMT5-dependent MYCN decrease in NGP cells suggests an alternative mechanism for PRMT5 compared to the BET domain inhibitor JQ-1, which did not affect MYCN in NGP cells (Puissant et al., 2013). MYCN transcript levels were only moderately decreased accompanying PRMT5 knockdown (Figure S4B), suggesting that PRMT5 mediates MYCN regulation at post-transcriptional levels, although we cannot currently exclude composite effects including indirect transcriptional regulation, for example by PRMT5 depletion leading to reactivation of epigenetically silenced MYCN repressor proteins or microRNAs.


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)

MYCN depletion triggered by PRMT5 knockdown. (A) Immunoblot analysis showing MYCN depletion in SK-N-BE(2)C, NGP and Kelly NB cell-lines following transfection with two independent PRMT5 siRNAs, negating the possibility of off-target effects. (B) PRMT5 knockdown in the SHEP-Tet21N cell-line harbouring inducible MYCN expression. MYCN is switched off in the presence of tetracycline (+Tet) and induced after its removal (−Tet). Cell death is clearly visible in the MYCN-on cells after PRMT5 knockdown. (C) Cell counts showing significantly increased cell death (asterisked, P < 0.005, Student's t-test) after PRMT5 knockdown in cells induced to express MYCN. A statistically insignificant change (not significant, ns) is observed in MYCN-off cells. (D) Immunoblots of SHEP-Tet21N cells demonstrating that PRMT5 also affects MYCN expressed from the inducible transgene. Depletion is accompanied by an increase in cleaved PARP indicative of apoptosis.
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fig4: MYCN depletion triggered by PRMT5 knockdown. (A) Immunoblot analysis showing MYCN depletion in SK-N-BE(2)C, NGP and Kelly NB cell-lines following transfection with two independent PRMT5 siRNAs, negating the possibility of off-target effects. (B) PRMT5 knockdown in the SHEP-Tet21N cell-line harbouring inducible MYCN expression. MYCN is switched off in the presence of tetracycline (+Tet) and induced after its removal (−Tet). Cell death is clearly visible in the MYCN-on cells after PRMT5 knockdown. (C) Cell counts showing significantly increased cell death (asterisked, P < 0.005, Student's t-test) after PRMT5 knockdown in cells induced to express MYCN. A statistically insignificant change (not significant, ns) is observed in MYCN-off cells. (D) Immunoblots of SHEP-Tet21N cells demonstrating that PRMT5 also affects MYCN expressed from the inducible transgene. Depletion is accompanied by an increase in cleaved PARP indicative of apoptosis.
Mentions: To further investigate the strong association between PRMT5 and MYCN status, we next examined the effect of PRMT5 knockdown on MYCN in SK-N-BE(2)C, NGP and Kelly cell-lines, all of which have MYCN amplification. As shown in Figure 4A, PRMT5 depletion was accompanied by a dramatic decrease of MYCN protein with two independent PRMT5 siRNAs, thereby negating the possibility of off-target effects. Notably, the PRMT5-dependent MYCN decrease in NGP cells suggests an alternative mechanism for PRMT5 compared to the BET domain inhibitor JQ-1, which did not affect MYCN in NGP cells (Puissant et al., 2013). MYCN transcript levels were only moderately decreased accompanying PRMT5 knockdown (Figure S4B), suggesting that PRMT5 mediates MYCN regulation at post-transcriptional levels, although we cannot currently exclude composite effects including indirect transcriptional regulation, for example by PRMT5 depletion leading to reactivation of epigenetically silenced MYCN repressor proteins or microRNAs.

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