Fig. 9

ARMC5 with mutations found in the MM and PBMAH patients had compromised affinity and function. A A schematic illustrating the ARMC5 protein structure with mutations (red dots) found in MM patients. The upper schematic shows the most abundant 935-aa ARMC5 isoform, and the mutation positions are numbered according to this isoform. The lower schematic shows the longest 1030-aa isoform, with 95 extra aa in the N-terminus. The R429C mutation in this long isoform is the same as the R334C mutation in the 935-aa isoform. B Reduced binding between ARMC5 R334C and POLR2A in HEK293 cells. HEK293 cells were transfected with plasmids expressing WT ARMC5-HA or ARMC5 R334C mutant-HA. The association of WT and mutant ARMC5 with endogenous POLR2A was detected according to anti-HA Ab immunoprecipitation followed by anti-POLR2A Ab (4H8) immunoblotting. C Reduced binding of ARMC5 R793Q, P559L, and G422S with POLR2A in HEK293 cells. HEK293 cells were transfected with constructs expressing FLAG-tagged WT or mutant ARMC5s. An empty vector was used as an additional control. The association of WT and mutant ARMC5 with endogenous POLR2A was detected according to anti-FLAG Ab immunoprecipitation followed by anti-POLR2A Ab (F12) immunoblotting. D Reduced binding between ARMC5 R793Q mutant and endogenous CUL3. HEK293 cells were transfected with constructs expressing FLAG-tagged WT ARMC5 or ARMC5 R793Q mutant. The association of WT or mutant ARMC5 with the endogenous CUL3 was detected according to anti-FLAG Ab immunoprecipitation followed by anti-CUL3 Ab immunoblotting. E A volcano plot showing reduced binding between the FLAG-ARMC5 R315W mutant (a mutation found in PBMAH patients) expressed in HEK293 cells and POLR2A according to anti-FLAG Ab immunoprecipitation followed by LC-MS/MS. The horizontal line: FRD = 0.05, based on three biological replicates. The vertical lines: + 2- and − twofold changes. F Reduced POLR2A ubiquitination in HEK293 cells expressing 4 mutant ARMC5s. HEK293 cells were transfected with constructs expressing FLAG-tagged WT or mutant ARMC5s along with plasmid expressing ubiquitin-HA. An empty vector was used as an additional control. Ubiquitinated POLR2A was detected by anti-HA precipitation followed by anti-POLR2A Ab (4H8) blotting. Signals of all ubiquitinated proteins in the precipitates (second row) were used to monitor the immunoprecipitation efficiency. The signals of ARMC5 (detected by anti-FLAG Ab) and α-actinin in the lysates were used to confirm similar ARMC5-FLAG expression and input protein quantity, respectively, in each sample. Bar graphs in B and F summarize the results from more than 3 repetitions, using the ratios of densitometry signals of POLR2A (B) or ubiquitinated POLR2A (F) versus their respective controls (ARMC5 for B and Ub-HA for F). *p < 0.05; **p < 0.01 (paired two-way Student’s t tests)