Figure 5.

Post-Translational Modification by Arginine Methylation Alters the Phase Transition of Ddx4^N1

(A) Sequence logo (weblogo.berkeley.edu) depicting the amino acid motifs surrounding arginine residues of Ddx4^N1 predominantly targeted by PRMT1. Arginine residues to be converted to aDMA are highlighted in dark red and with two small ellipses. The amino acid numbers of the modified arginine residues are shown within their respective sequence contexts. Asterisks highlight aDMA sites identified in Ddx4^N1Me with 95% probability (Scaffold score) from a combination of trypsin and GluC digestion of recombinant, purified Ddx4^N1Me. aDMA at sites 146 and 147 was identified at ∼65% probability (Scaffold score).

(B) Schematic and mass reconstruction of +TOF MS spectra of Ddx4^N1 (green; 25.833 kDa) and Ddx4^N1Me (dark red). In the latter, a series of peaks was observed between 1 and 20 methyl additions. The major peaks indicate complete aDMA modification at 5 and 6 sites, respectively.

(C) A schematic of aDMA together with an insert showing the ¹H-¹³C HSQC NMR spectrum of the ^θCH₃ of Ddx4^N1Me. The chemical shifts of the methyl groups verify that the modification is aDMA (see Figure S5).

(D) The phase-transition temperatures of Ddx4^N1Me (dark red) are shifted compared to the unmodified form under the same conditions (light green). Modification with aDMA at a mixture of 5–6 aDMA sites reduces the transition temperature by 25°C, an effect on the phase transition comparable to increasing the ionic strength by 100 mM.