Rtners or substrates of PRMT6. Some of the identified PRMT6’s partners support the reliability of our Y2H screening. hnRNP Q and snRNPB are already known to be substrates for PRMTs. Med28 and MTF2 are subunits of macromolecular complexes involved in gene transcription and chromatin epigenetic modulation, activities that have been firmly established also for PRMT6 [6?]. hnRNP Q has been previously shown to be 13655-52-2 site methylated in vitro by PRMT1 and its in vivo methylation is important for its nuclear localization [26] and for insulin receptor trafficking and insulin signalling [27]. The small nuclear ribonucleoprotein B and B1 (snRNPB), which is involved in several steps of 
the biogenesis of the snRNPs, has also been found methylated on arginine residues but the PRMT responsible for this modification has not been identified yet [28]. Med28 is a subunit of Mediator, a transcriptional cofactor that regulates transcription of nearly all RNA polymerase II-dependent genes [29] while MTF2 is a member of polycomb repressive complex-2 (PRC2) that is involved in the epigenetic regulation of a large number of genes [30].Mapping the PRMT6’s Protein-protein Interaction DomainLittle information is available about the domain organization of PRMT6 and PRMTs in general, therefore the region of PRMT6 involved in the interaction with the putative partners was mapped. To this end five different N- and C-terminal deletion mutants of PRMT6 were generated (Fig. 1A). Before testing the interaction in Y2H assay, their expression in yeast was assessed by western blot.Figure 3. In vivo confirming PRMT6’s partners by Co-Affinity purification (Co-AP). (A) PRMT6 fused to Maltose Binding Protein (MBP-PRMT6) or Maltose Binding Protein (MBP) alone were produced by transient transfection in HEK293T cells. Cellular lysates were LY-2409021 custom synthesis incubated with amylose resin and affinity captured MBP-PRMT6 and MBP proteins recovered. Proteins were separated by SDS-PAGE (T = 10 ) and analysed by WB using an a-HA antibody. Lanes 1 and 3: input, 5 of the amount used; lanes 2 and 4: co-affinity purified proteins. Experiments were repeated at least twice and a representative result is shown. (B) The ponceau stained membrane of a representative experiment is shown. doi:10.1371/journal.pone.0053750.gmembrane-bounded organelle (Table S2). From the gene ontology (GO) annotations of the identified partners, PRMT6 appears to be involved in protein complex assembly, RNA processing, and regulation of protein metabolic processes, in particular nucleoside and nucleotide metabolic processes. None of the proteins listedFigure 4. Discovering new substrates for PRMT6. (A) Recombinant PRMT6 partners in fusion with GST were incubated with radiolabelled S-adenosyl-L-(methyl-3H)-methionine and GST-PRMT6 for in vitro methylation assay (lanes 2?, 8, 9). GST and GST-GAR (lanes 1 and 7) were used as negative and positive control, respectively. Proteins were separated by SDS-PAGE (T = 10 ) and checked by fluorography. Experiments were repeated at least twice and a representative result is shown. (B) Blue Comassie staining was used to check both the correct production and the amount of recombinant proteins. Arrows indicate the position of PRMT6. doi:10.1371/journal.pone.0053750.gThe Protein-Protein Molecular Network of PRMTThey were all correctly expressed except mutant PRMT6 87?84 (Fig. 1B). Each PRMT6 deletion mutant was tested for the interaction with a subset of 31 partners out of the 36 identified. Full length PRMT6 an.Rtners or substrates of PRMT6. Some of the identified PRMT6’s partners support the reliability of our Y2H screening. hnRNP Q and snRNPB are already known to be substrates for PRMTs. Med28 and MTF2 are subunits of macromolecular complexes involved in gene transcription and chromatin epigenetic modulation, activities that have been firmly established also for PRMT6 [6?]. hnRNP Q has been previously shown to be methylated in vitro by PRMT1 and its in vivo methylation is important for its nuclear localization [26] and for insulin receptor trafficking and insulin signalling [27]. The small nuclear ribonucleoprotein B and B1 (snRNPB), which is involved in several steps of the biogenesis of the snRNPs, has also been found methylated on arginine residues but the PRMT responsible for this modification has not been identified yet [28]. Med28 is a subunit of Mediator, a transcriptional cofactor that regulates transcription of nearly all RNA polymerase II-dependent genes [29] while MTF2 is a member of polycomb repressive complex-2 (PRC2) that is involved in the epigenetic regulation of a large number of genes [30].Mapping the PRMT6’s Protein-protein Interaction DomainLittle information is available about the domain organization of PRMT6 and PRMTs in general, therefore the region of PRMT6 involved in the interaction with the putative partners was mapped. To this end five different N- and C-terminal deletion mutants of PRMT6 were generated (Fig. 1A). Before testing the interaction in Y2H assay, their expression in yeast was assessed by western blot.Figure 3. In vivo confirming PRMT6’s partners by Co-Affinity purification (Co-AP). (A) PRMT6 fused to Maltose Binding Protein (MBP-PRMT6) or Maltose Binding Protein (MBP) alone were produced by transient transfection in HEK293T cells. Cellular lysates were incubated with amylose resin and affinity captured MBP-PRMT6 and MBP proteins recovered. Proteins were separated by SDS-PAGE (T = 10 ) and analysed by WB using an a-HA antibody. Lanes 1 and 3: input, 5 of the amount used; lanes 2 and 4: co-affinity purified proteins. Experiments were repeated at least twice and a representative result is shown. (B) The ponceau stained membrane of a representative experiment is shown. doi:10.1371/journal.pone.0053750.gmembrane-bounded organelle (Table S2). From the gene ontology (GO) annotations of the identified partners, PRMT6 appears to be involved in protein complex assembly, RNA processing, and regulation of protein metabolic processes, in particular nucleoside and nucleotide metabolic processes. None of the proteins listedFigure 4. Discovering new substrates for PRMT6. (A) Recombinant PRMT6 partners in fusion with GST were 
incubated with radiolabelled S-adenosyl-L-(methyl-3H)-methionine and GST-PRMT6 for in vitro methylation assay (lanes 2?, 8, 9). GST and GST-GAR (lanes 1 and 7) were used as negative and positive control, respectively. Proteins were separated by SDS-PAGE (T = 10 ) and checked by fluorography. Experiments were repeated at least twice and a representative result is shown. (B) Blue Comassie staining was used to check both the correct production and the amount of recombinant proteins. Arrows indicate the position of PRMT6. doi:10.1371/journal.pone.0053750.gThe Protein-Protein Molecular Network of PRMTThey were all correctly expressed except mutant PRMT6 87?84 (Fig. 1B). Each PRMT6 deletion mutant was tested for the interaction with a subset of 31 partners out of the 36 identified. Full length PRMT6 an.