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. 2024 May 6;52(12):7225–7244. doi: 10.1093/nar/gkae319

Figure 2.

Figure 2.

PRMT5-mediated arginine methylation promotes PTM of FXR1. (A) The protein structure of FXR1 protein has regions marked for its different domains. The C-terminal arginine-glycine-glycine (RGG) RNA-binding domain has the methylated arginine (R) residues marked in the illustration. (B) Multiple sequence alignment of the C-terminus of human and mouse FXR1 and FMRP proteins is shown. Secondary structural elements are marked above the sequences, with α-helices depicted as cylinders and β-strands as arrows. The R residues potentially methylated inside the cell have been chosen for the mutation to lysine (K) and are highlighted (yellow). The FXR1 residue numbers are given above the sequence. The numbers in parentheses indicate the length of the sequences shown. (C) Immunoblot analyses of WT and mutant Myc-FXR1 protein expressions in HEK293T cells are shown. β-Actin serves as a loading control. (D) HEK293 cells expressing empty vector and Myc-tag FXR1 (WT) were used for IP with Myc-tag antibody and probed for SDMA, ADMA and Myc-tag antibodies. The empty vector serves as a control for Myc-FXR1. (E) HEK293 cells expressing empty vector, Myc-tag FXR1 (WT), and mutant (R386-459K) were used for IP with Myc-tag antibody and probed for SDMA and Myc-tag antibodies. (F) HEK293 cells expressing empty vector, Myc-tag FXR1 (WT), and mutants R386K, R388K, R453K, R455K and R459K were used for IP with Myc-tag antibody and probed for SDMA, PRMT5, PRMT1 and FMRP (positive control). The bottom panel depicts the quantitative value of WT and RGG mutants FXR1 protein interaction with PRMT5. N = 3. (G) A549 cells stably expressing Myc-tag FXR1 (WT) and mutant (R386-459K) were treated with 5 μM cycloheximide treatment for 0 to 10 h to block protein synthesis. After the treatment, the cells were harvested at the indicated time points and immunoblotted for FXR1 and β-actin (loading control). The bottom graph shows the relative FXR1 protein levels with time after cycloheximide treatment. All the data were defined as mean ± SD and were analyzed by Student's t-test (n = 3). *P < 0.05.