Regulation of mitochondrial gene expression, RNA binding, and gRNP complex formation by arginine methylation.

摘要

Trypanosoma brucei is a parasitic protozoan and the causative agent of African sleeping sickness. T. brucei contains a single, large mitochondrion whose biologic functions are rapidly and drastically altered with environmental changes and life cycle stages. RBP16 is an abundant RNA-binding protein present in T. brucei mitochondria. Previous studies demonstrated that RBP16 is involved in multiple aspects of T. brucei mitochondrial gene regulation including RNA stability and editing. Multiple residues in the arginine/glycine-rich C-terminus of RBP16 have been found to be post-translationally modified through arginine (Arg) methylation. Arginine methylation is a broad regulator of protein function and could affect RBP16 function and its capacity to regulate T. brucei mitochondrial gene expression.; In Chapter II, experiments were performed that identified arginine methylation as a key point of regulation for RBP16 function and as a specific modulator of T. brucei mitochondrial gene expression. The analysis of cells over-expressing a non-methylatable RBP16 mutant identified RBP16 as an effector of arginine methylation action in T. brucei mitochondria, and showed that arginine methylation is a key point of regulation for RBP16 function. Further effects due to depletion of the major type I methyltransferase in T. brucei, TbPRMT1, were identified that cannot be explained by RBP16 action, thereby implicating additional unidentified TbPRMT1 substrates in the regulation of mitochondrial gene expression.; In Chapter III, the major RBP16 containing ribonucleoprotein (RNP) complexes were identified while determining a role for Arg methylation in their formation. It was demonstrated that native RBP16 forms two endogenous complexes of ∼5S and 11S and that the 11S complex contains a guide RNA (gRNA) component. Arg methylation, catalyzed by TbPRMT1, is required to maintain the RBP16-protein interactions that mediate 5S and 11S complex assembly and/or stability. At the same time, this modification decreases the ability of RBP16 to associate with gRNA and increases its association with several mRNAs. Thus, Arg methylation has specific effects on RBP16 gRNA and mRNA association and gRNP formation.; Through these studies, arginine methylation has emerged as a broad regulator of mitochondrial gene expression through the modulation of RNA stability, RNA binding, and protein-protein interactions involving the mitochondrial protein RBP16.