Figure 1.
PRMT6 protein-protein interaction domain maps mainly to the N-terminal protein portion 1–86.
(A) Schematic representation of the PRMT6 deletion mutants. The central region containing the catalytic domain is indicated in black. Numbers correspond to the aminoacids positions. Positive interactions out of 31 partners tested are indicated on the right. (B) Total protein extracts from yeast expressing the different PRMT6 deletion mutants in fusion with LexA DNA binding domain, grown in medium containing glucose (Glu) or galactose and raffinose (Gal/Raf), were separated in SDS-PAGE (T = 10%) and analyzed by western blot using an α-LexA antibody. Molecular weights are indicated on the left.
Figure 2.
Confirming PRMT6’s partners and extending HMGA1a molecular network by a hypothesis driven approach.
(A) Putative PRMT6 molecular partners were in vitro translated (IVT) and radiolabelled by incorporation of 35S-methionine. GST pull-down assays were performed with GST, GST-PRMT6, GST-HMGA1b, and GST-HMGA2. For each partner, in lane 1 (input) a quantity corresponding to 20% of the IVT used in the GST pull-down experiments was loaded. Proteins are visualized by fluorography. Experiments were repeated at least twice and a representative result is shown. (B) A representative SDS-PAGE (T = 10%) of the proteins used in the experiments, stained with Blue Comassie, is shown.
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 α-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.
Figure 4.
Discovering new substrates for PRMT6.
(A) Recombinant PRMT6 partners in fusion with GST were incubated with radio-labelled S-adenosyl-L-(methyl-3H)-methionine and GST-PRMT6 for in vitro methylation assay (lanes 2–6, 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.
Figure 5.
HMGA1a modulates the methyltransferase activity of PRMT6 toward MIF.
(A) Constant amount of GST-MIF were incubated with recombinant GST-PRMT6 and radio-labelled S-adenosyl-L-(methyl-3H) methionine in the presence of increasing amounts of full length HMGA1 (FL), a truncated HMGA1a form (1–51), and a HMGA1a mutated form (R57,59A) for in vitro methylation assays. Methylation reactions of MIF and HMGA1a alone represent control experiments. Proteins were separated by SDS-PAGE (T = 15%) and checked by fluorography. Experiments were repeated at least twice and a representative result is shown. (B) Blue Comassie staining was used to check for the quantification of HMGA1a proteins. (C) Schematic representation of the FL, 1–51 and R57,59A HMGA1a domain organization.
Figure 6.
From interaction data to functional hypothesis.
Schematic representation of the molecular network impinging on p53 and directly linked to PRMT6. The modulatory effects reported in this scheme (activation or blocking) are exclusively mediated by direct protein-protein interactions. The methyltransferase activity of PRMT6 toward its substrates implies a direct protein-protein contact.
Table 1.
Y2H 1st: yeast two-hybrid screening.