Effects of Asymmetric Arginine Dimethylation on RNA‐Binding Peptides

Arginine is the most frequent methylation site (about 90 %) in proteins found in higher eukaryotes. Numerous studies have shown that Arg methylation in RNA-binding proteins (RBP) can regulate pre-mRNA processing and control the stability of mRNA through modulation of both protein–protein and protein–RNA interactions. Many RBP’s, as well as proteins involved in transcription and RNA metabolism, serve as substrates for the enzyme Arg methyl transferase (PRMT). Since Arg methylation can be reversed by enzyme catalyzed demethylation, the reversible modification plays a critical role as a gene-control mechanism. The role(s) of Arg methylation of proteins, however, remains an intriguing question, especially with regard to molecular recognition of RNA, even though it has both positive and negative effects on protein–protein interactions. The results of previous studies with a small number of synthetic Arg methylated peptides led to the conclusion that methylation does not promote significant changes in binding affinities to RNA. Also, in biological experiments, no significant physiological changes or reductions 10] in binding affinities with RNA were observed upon Arg methylation. However, these investigations employed indirect methods, utilized random sequences from the repeated sequence Arg-Gly-Gly found in nuclear and hnRNP proteins (RGG box), and involved comparisons of properties of the native proteins with those of Lys and Ala mutants. 10] Consequently, the findings and conclusions might have misrepresented the role that methylation plays in interactions between proteins and RNA. To analyze the physicochemical changes that arise from alterations in the affinities of methylated proteins (or peptides) for their cis-element RNA, we thought that a large number of modified proteins (or peptides) should be probed. The Rev peptide was chosen as a model for this purpose, because it contains ten Arg residues in its RNA-binding motif. The well known RBP, Rev, is a key peptide in HIV propagation. When ACHTUNGTRENNUNGunspliced or partially spliced viral RNA is imported into the nucleus, Rev binds to the Rev responsive element (RRE) in the RNA of HIV-1 and increases gene transcription. Furthermore, the Rev peptide has a-helical structures, which provide opportunities to explore the effects of methylation on conformational rigidity. The effects of methylation might depend on the position and number of asymmetric dimethylated Arg residues, which result from the operation of a major methylation mode. 12] A recent report suggested that Rev is a substrate for asymmetric dimethylation. But physicochemical effects of methylation on the individual Arg residues have not been demonstrated yet. In this study, all possible Rev mutant peptides containing asymmetric dimethylated Arg residues were synthesized and their binding affinities to RRE were measured. The results, described below, demonstrate that a variety of binding affinity changes take place upon methylation of the Arg residues of Rev; these range from a 25-fold reduction to a 1.3-fold increase in binding affinity to RRE RNA. The findings suggest that the affinity changes brought about by methylation are highly ACHTUNGTRENNUNGdependent on the position of the Arg residue, and lead to the interesting conclusion that post-translational methylation could be used process as a reversible switch to differentiate protein–RNA interactions in a selective manner. A library of peptides was synthesized by using the standard Fmoc solid-phase protocol. The dissociation constant (Kd) for the binding of each peptide to RRE was measured by using the electrophoretic gel mobility shift assay (Figure 1, Table 1). As expected, modifications of Arg residues that have been identified to have the most contact with RRE based on the so-