The Protein Arginine Methyltransferase 6 (PRMT6) is an enzyme characterized by a predominant nuclear localization and automethylation activity. Until today, very few information are known about its function and its substrates. To better characterize PRMT6, we looked for new partners and substrates, using the yeast two-hybrid system. This technique allowed us to discover 36 new partners for this enzyme, 19 of these interactions were confirmed with the GST Pull-down assay. Among these partners, 9 resulted to be interactors also for HMGA protein, a chromatin architectural factor that has been previously demonstrated to be an in vivo substrate of PRMT6. The binding between partners and PRMT6 was further assessed in vivo in mammalian cells, and out of nine proteins tested, 7 were confirmed. To test whether among the identified partners there were substrates, we performed an in vitro methylation assay, and discovered 4 new substrates of this enzyme (Macrophage migration inhibitory factor, Human DnaJ homologue, Small nuclear ribonucleoprotein-associated proteins B and B', and Tubulin beta-2A chain). Moreover, we evaluated if the presence of HMGA1a, could modulate the methylation activity of PRMT6. We demonstrated that MIF's methylation was enhanced in the presence of HMGA1a protein. Furthermore, to evaluate a possible role played by HMGA, MIF, and PRMT6 together, we studied the effect of MIF on Cyclin A (CycA) promoter. We choose this reporter because it is known the activation effect of HMGA2 protein on this promoter. Our data demonstrated that both MIF and PRMT6 could activate this promoter, but the simultaneous presence of these two proteins result in a lower activation, suggesting that PRMT6 had a negative effect on MIF function.

PRMT6 : identification and characterization of new molecular partners

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2010

Abstract

The Protein Arginine Methyltransferase 6 (PRMT6) is an enzyme characterized by a predominant nuclear localization and automethylation activity. Until today, very few information are known about its function and its substrates. To better characterize PRMT6, we looked for new partners and substrates, using the yeast two-hybrid system. This technique allowed us to discover 36 new partners for this enzyme, 19 of these interactions were confirmed with the GST Pull-down assay. Among these partners, 9 resulted to be interactors also for HMGA protein, a chromatin architectural factor that has been previously demonstrated to be an in vivo substrate of PRMT6. The binding between partners and PRMT6 was further assessed in vivo in mammalian cells, and out of nine proteins tested, 7 were confirmed. To test whether among the identified partners there were substrates, we performed an in vitro methylation assay, and discovered 4 new substrates of this enzyme (Macrophage migration inhibitory factor, Human DnaJ homologue, Small nuclear ribonucleoprotein-associated proteins B and B', and Tubulin beta-2A chain). Moreover, we evaluated if the presence of HMGA1a, could modulate the methylation activity of PRMT6. We demonstrated that MIF's methylation was enhanced in the presence of HMGA1a protein. Furthermore, to evaluate a possible role played by HMGA, MIF, and PRMT6 together, we studied the effect of MIF on Cyclin A (CycA) promoter. We choose this reporter because it is known the activation effect of HMGA2 protein on this promoter. Our data demonstrated that both MIF and PRMT6 could activate this promoter, but the simultaneous presence of these two proteins result in a lower activation, suggesting that PRMT6 had a negative effect on MIF function.
2010
en
Cromatina
HMGA
Interattoma
Metilazione
Prmt6
Ptms
SCUOLA DI DOTTORATO DI RICERCA IN BIOMEDICINA MOLECOLARE
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.14242/272371
Il codice NBN di questa tesi è URN:NBN:IT:UNITS-272371