Fig 1: Model. The interaction between NCL and G4 of EBNA1 mRNA is direct and the C-terminal RGG motif of NCL is required for both the ability of NCL to interact with G4 that form in the GAr-encoding sequence of EBNA1 mRNA and for NCL’s crucial role in GAr-based inhibition of translation, the molecular mechanism at the root of EBNA1 immune evasion. RGG motifs are the main substrates of type I PRMTs. The interaction between NCL and G4 of EBNA1 mRNA, as well as the GAr-based inhibition of translation and EBNA1 immune evasion, depend on type I PRMTs, in particular PRMT1. Results of the site-directed mutagenesis of the arginines of the RGG motif of NCL experiments suggest that the role of type I PRMTs in EBNA1 immune evasion involves methylation of the arginines of the RGG motif of NCL. Hence, two possible models for the role RGG methylation by type I PRMTs in EBNA1 evasion can be envisaged: direct (A) or indirect (B). In the direct model (A), the methylation of arginines of the RGG motif of NCL would directly favour the interaction of the latter with G4 of EBNA1 mRNA and therefore the inhibition of its translation, which in turn allows EBNA1 and EBV to evade the immune system. In the indirect model (B), the non-methylated RGG motif of NCL would be sequestered by a protein partner [protein X, which may be nucleolin itself or another protein such as the various ribosomal proteins which have been shown to interact with the RGG motif of NCL (62)], thereby preventing its interaction with G4 of EBNA1 mRNA. In this model, methylation of the arginines of the RGG motif would interfere with its interaction with protein X, thus releasing NCL and allowing its RGG motif to interact with G4 of EBNA1 mRNA. Our in vitro assays did not show any significant difference between methylated and unmethylated RGG for the binding of EBNA1 mRNA G4, suggesting the indirect role is the most likely.
Fig 2: Crosstalk between PRMT1-catalyzed arginine methylation and p38α-mediated phosphorylation of TDP-43(A) Western blot of immunoprecipitated FLAG-tagged TDP-43 from SH-SY5Y cells probed with antibodies against TDP-43, pTDP-43, and MMA. GAPDH serves as a loading control. The positions of FLAG-tagged TDP-43 (exo) and endogenous TDP-43 (endo) are indicated.(B) Western blot of immunoprecipitated FLAG-tagged TDP-43WT from SH-SY5Y cells with or without siRNA-induced p38α knockdown probed with antibodies against TDP-43, p38α, pTDP-43, and MMA. GAPDH serves as a loading control.(C) Quantification of TDP-43-CTF/full length-ratio (mean ± SD, unpaired t test, n = 3). **p < 0.01.(D) Quantification of mono-methylated TDP-43-CTF normalized to total TDP-43-CTF (mean ± SD, unpaired t test, n = 3). *p < 0.05.(E) Western blot of immunoprecipitated FLAG-tagged TDP-43WT from SH-SY5Y cells with or without PRMT1 overexpression probed with antibodies against TDP-43, pTDP-43, and MMA. GAPDH serves as a loading control. The positions of FLAG-tagged TDP-43 (exo) and endogenous TDP-43 (endo) are indicated. The positions of Myc-tagged PRMT1 (exo) and endogenous PRMT1 (endo) are also indicated. See also Figure S7.
Fig 3: TDP-43 is methylated by PRMT1 at R293(A) Sequence alignment of amino acids 287–307 of TDP-43 from diverse vertebrates. Conserved 292–293 sites are bolded. S292 phosphorylation site and R293-G295 RGG-motif are highlighted in yellow and green, respectively.(B) Western blot of immunoprecipitated endogenous TDP-43 from SH-SY5Y-cells probed with antibodies against TDP-43, ADMA, MMA, and SDMA. TDP-43 with arginine methylation is marked by arrowheads.(C) Western blot of immunoprecipitated endogenous TDP-43 from SH-SY5Y-cells with siRNA-induced PRMT1 knockdown probed with antibodies against TDP-43 or MMA. A PRMT1 blot was run separately to confirm siRNA-mediated knockdown of PRMT1.(D) Western blot of expressed and immunoprecipitated TDP-43WT using anti-FLAG antibody from SH-SY5Y-cells treated with DMSO (D) or with methyltransferase inhibitor AdOx (A) at a final concentration of 20 μM for 24 h. FL, full-length TDP-43; CTF35, C-terminal 35-kDa fragment of TDP-43.(E) Western blot of expressed and immunoprecipitated TDP-43WT and TDP-43R293K using anti-FLAG antibody from SH-SY5Y cells probed with antibodies against TDP-43 and MMA. See also Figures S6 and S7.
Supplier Page from Abcam for Recombinant human PRMT1 protein (His tag N-Terminus + MBP tag N-Terminus)