D ornitine decarboxylase (ODC) were included as positive and negative control, respectively. Results reported in Table S3 and summarized in Fig. 1A show that 27 out of 31 partners tested are able to interact with the deletion mutant PRMT6 1?6, while none of them is able to bind mutants lacking the N-terminal portion of PRMT6, suggesting therefore that the N-terminal region is necessary and sufficient for the MedChemExpress AKT inhibitor 2 association with the partners. It is worthwhile noting that, in a first attempt to clone PRMT6 a cDNA was isolated encoding for a 60 aa Nterminal truncated form of PRMT6 that was missing methyltransferase activity [5], therefore suggesting an involvement of this region in substrate docking. Further evidences suggest a role for the N-terminal region of PRMTs in substrate binding order ZK 36374 specificity and enzymatic activity. Indeed, it has been demonstrated that alternative splicing of PRMT1 generates several N-terminal isoforms differing in catalytic activity and substrate specificity [31] and that N-terminal domain of PRMT8 modulates its activity [32]. These observations are supported by data evidencing that within PRMT1 substrates, positively charged residues distal to the modified arginine are involved in the process of docking to the enzyme surface [33]. In agreement with this, a surface scanning mutational analysis of PRMT1 revealed that mutation of Nterminal acidic residues within the EEMxxD motif strongly impairs substrate binding [34]. It is noteworthy to evidence that this 
motif is perfectly conserved between PRMT1 and PRMT6. Previously structural work on PRMT1, 3 and 5 [35?7] suggested a role for the N-terminal portion of PRMTs in proteinprotein interactions and the possibility that the sequence variability of PRMTs could be responsible for their different substrate specificity. All these structural and biochemical data suggest a key role of the N-terminal portion of PRMTs in substrate binding and are in agreement with our data demonstrating that the N-terminal portion of PRMT6 (aa 1?6) is essential for binding to its molecular partners.Since PRMT6 is a HMGA interactor and a histone modifier that can be part of DNA-bound complexes, we hypothesized that HMGA and PRMT6 could have overlapping molecular contexts and common partners. Therefore, PRMT6 partners were assayed in GST-pull down experiments for their interaction with two HMGA proteins: HMGA1b (the shorter isoform of HMGA1 proteins), and HMGA2. HMGA1a isoform turned out not to be efficiently produced as a GST-fusion product (data not shown). In agreement with our hypothesis, among the 19 confirmed PRMT6’s interactors, 9 were found to interact with both HMGA1b and HMGA2 (hnRNP Q, snRNPB, PRPF39, MIF, PTPS, COPS3, CASP6, SVEP1 and HSJ-2) and 1 protein (RNA binding protein NOB1) resulted to specifically interact only with HMGA2. To further validate our screening, 9 partners were selected to be assayed in vivo for their ability to bind PRMT6 using co-Affinity Purification (co-AP). PRMT6 was cloned in fusion with the Maltose Binding protein (MBP) and co-transfected in cells together 16574785 with partners cloned in fusion with HA tag. Cells were lysed in native conditions, the complexes were purified with the amylase resin, analysed by SDS PAGE, and the partners detected by western blot analyses using a-HA antibody. Also in this case, the vast majority of the tested proteins turned out to be confirmed. Indeed, Fig. 3 shows that among the partners tested, 7 (MTF2, Nm23-H1, NOB1, PTPS, CASP6, TU.D ornitine decarboxylase (ODC) were included as positive and negative control, respectively. Results reported in Table S3 and summarized in Fig. 1A show that 27 out of 31 partners tested are able to interact with the deletion mutant PRMT6 1?6, while none of them is able to bind mutants lacking the N-terminal portion of PRMT6, suggesting therefore that the N-terminal region is necessary and sufficient for the association with the partners. It is worthwhile noting that, in a first attempt to clone PRMT6 a cDNA was isolated encoding for a 60 aa Nterminal truncated form of PRMT6 that was missing methyltransferase activity [5], therefore suggesting an involvement of this region in substrate docking. Further evidences suggest a role for the N-terminal region of PRMTs in substrate binding specificity and enzymatic activity. Indeed, it has been demonstrated that alternative splicing of PRMT1 generates several N-terminal isoforms differing in catalytic activity and substrate specificity [31] and that N-terminal domain of PRMT8 modulates its activity [32]. These observations are supported by data evidencing that within PRMT1 substrates, positively charged residues distal to the modified arginine are involved in the process of docking to the enzyme surface [33]. In agreement with this, a surface scanning mutational analysis of PRMT1 revealed that mutation of Nterminal acidic residues within the EEMxxD motif strongly impairs substrate binding [34]. It is noteworthy to evidence that this motif is perfectly conserved between PRMT1 and PRMT6. Previously structural work on PRMT1, 3 and 5 [35?7] suggested a role for the N-terminal portion of PRMTs in proteinprotein interactions and the possibility that the sequence variability of PRMTs could be responsible for their different substrate specificity. All these structural and biochemical data suggest a key role of the N-terminal portion of PRMTs in substrate binding and are in agreement with our data demonstrating that the N-terminal portion of PRMT6 (aa 1?6) is essential for binding to its molecular partners.Since PRMT6 is 
a HMGA interactor and a histone modifier that can be part of DNA-bound complexes, we hypothesized that HMGA and PRMT6 could have overlapping molecular contexts and common partners. Therefore, PRMT6 partners were assayed in GST-pull down experiments for their interaction with two HMGA proteins: HMGA1b (the shorter isoform of HMGA1 proteins), and HMGA2. HMGA1a isoform turned out not to be efficiently produced as a GST-fusion product (data not shown). In agreement with our hypothesis, among the 19 confirmed PRMT6’s interactors, 9 were found to interact with both HMGA1b and HMGA2 (hnRNP Q, snRNPB, PRPF39, MIF, PTPS, COPS3, CASP6, SVEP1 and HSJ-2) and 1 protein (RNA binding protein NOB1) resulted to specifically interact only with HMGA2. To further validate our screening, 9 partners were selected to be assayed in vivo for their ability to bind PRMT6 using co-Affinity Purification (co-AP). PRMT6 was cloned in fusion with the Maltose Binding protein (MBP) and co-transfected in cells together 16574785 with partners cloned in fusion with HA tag. Cells were lysed in native conditions, the complexes were purified with the amylase resin, analysed by SDS PAGE, and the partners detected by western blot analyses using a-HA antibody. Also in this case, the vast majority of the tested proteins turned out to be confirmed. Indeed, Fig. 3 shows that among the partners tested, 7 (MTF2, Nm23-H1, NOB1, PTPS, CASP6, TU.
