Proteomic identification and comparative analysis of asymmetrically arginine-methylated proteins in immortalized, young and senescent cells

Protein‐arginine methylation is one of the modifications that yields mono and dimethyl (asymmetric or symmetric) arginine residues in proteins. Previously, we found that asymmetric arginine methylation is decreased proportionately with a decrease of cell proliferation potential of cells, and such arginine methylation is greatest in immortalized cells, followed by normal young cells, and lowest in replicatively senescent cells. Using an asymmetric dimethyl‐arginine‐specific antibody, we identified arginine‐methylated proteins in these cell types by immunoprecipitation and 2‐D immunoblotting followed by MS. As a result, arginine methylation of chaperone molecules and RNA‐binding proteins was differentially regulated between immortalized or young cells and senescent cells. Immortalized cells had significantly higher levels of methyl‐accepting proteins, such as cleavage stimulation factor 2 (CstF2) and heterogenous nuclear ribonucleoprotein (hnRNP) R, than young cells. However, senescent cells contained hypomethylaed CstF2, hnRNP K, and chaperone containing TCP1 subunit 7, as well as decreased hnRNP R level. Further, significant reduction of arginine modification in CstF2 and chaperone containing TCP1 subunit 7 was observed in prematurely senescent fibroblasts, induced by treatment with adenosine dialdehyde, a transmethylation inhibitor, or subcytotoxic concentration of H2O2. These results suggest that asymmetric modification of RNA‐binding proteins and molecular chaperones plays an essential role in maintaining cell proliferation capability.