Differential effects of arginine methylation on RBP16 mRNA binding, guide RNA (gRNA) binding, and gRNA-containing ribonucleoprotein complex (gRNP) formation

Mitochondrial gene expression in Trypanosoma brucei involves the coordination of multiple events including polycistronic transcript cleavage, polyadenylation, RNA stability, and RNA editing. Arg methylation of RNA binding proteins has the potential to influence many of these processes via regulation of protein-protein and protein-RNA interactions. Here we demonstrate that Arg methylation differentially regulates the RNA binding capacity and macromolecular interactions of the mitochondrial gene regulatory protein, RBP16. We show that, in T. brucei mitochondria, RBP16 forms two major stable complexes: a 5 S multiprotein complex and an 11 S complex consisting of the 5 S complex associated with guide RNA (gRNA). Expression of a non-methylatable RBP16 mutant protein demonstrates that Arg methylation of RBP16 is required to maintain the protein-protein interactions necessary for assembly and/or stability of both complexes. Down-regulation of the major trypanosome type 1 protein arginine methyltransferase, TbPRMT1, disrupts formation of both the 5 and 11 S complexes, indicating that TbPRMT1-catalyzed methylation of RBP16 Arg-78 and Arg-85 is critical for complex formation. We also show that Arg methylation decreases the capacity of RBP16 to associate with gRNA. This is not a general effect on RBP16 RNA binding, however, since methylation conversely increases the association of the protein with mRNA. Thus, TbPRMT1-catalyzed Arg methylation has distinct effects on RBP16 gRNA and mRNA association and gRNA-containing ribonucleoprotein complex (gRNP) formation.