Functional and biochemical analysis of the mitochondrial RNA binding protein RBP16 and its protein binding partner,p22

To understand the biochemical mechanisms by which RBP16 executes its role(s) in trypanosome mitochondrial gene expression, it is critical to examine in detail the macromolecular interactions in which RBP16 participates. This work describes experiments that further define the RNA binding properties of RBP 16 to gain insight into the nature of the molecular interactions as well as RNA length and sequence requirements involved in RBP16-gRNA binding. These results demonstrated that electrostatic interactions do not play a prominent role in the binding of RBP16 to gRNAs. Detailed RNA binding studies employing gRNA deletion mutants indicated that gRNA structure is not a critical binding determinant for RBP 16. These experiments also confirmed that the gRNA U tail is a major determinant for RBP16 binding. The identification and characterization of RBP 16 protein binding partners also provides clues regarding the function of RBP16 on a macromolecular level. Presented here is evidence that RBP 16 interacts directly with the previously identified mitochondrial protein, p22, in an RNA independent manner. This interaction elicits a significant stimulation of RBP16's RNA binding activity, up to ∼10 fold. The involvement of the CSD and RGG domains of RBP16 in both protein-RNA and protein-protein interactions was also delineated. These results indicate that the CSD of RBP16 plays a primary role in mediating both protein-RNA and protein-protein interactions, while the RGG domain stimulates both the RNA and protein binding capacity of the CSD.; Recently, it was demonstrated that RBP16 is important for the regulation of both RNA editing and stability. Therefore, experiments aimed at elucidating the mechanism of RBP16 function in RNA editing were' performed. In vitro RNA editing assays were utilized to demonstrate that recombinant RBP16 significantly stimulates insertion editing of both CYb and A6 pre-mRNAs. The mechanism by which RBP16 stimulates in vitro insertion editing is at least two fold. First, RBP16 appears to stimulate editing through an effect on pre-mRNA cleavage. This effect does not require RBP16 CSD interaction with RNA. These experiments additionally support a second mechanism of editing enhancement subsequent to cleavage that is influenced by CSD-gRNA binding. Overall, these results support a role for RBP16 in the posttranscriptional regulation of T. brucei mitochondrial gene expression and suggest a novel mechanism by which RBP 16 may regulate RNA editing in vivo. (Abstract shortened by UMI.)...