Structural and Biochemical Characterization of Trypanosoma brucei MRB1590, a kRNA Editing Accessory Protein

The typical flow of genetic information involves DNA being transcribed into RNA, which is then translated into protein. However, in some organisms this pathway requires an extra step where the RNA is post-transcriptionally modified before it can be translated. This process is referred to as RNA editing and can involve the substitution or the insertion and/or deletion of bases. Insertion/deletion RNA editing is rare, only occurring in the mitochondria of Physarum and trypanosomes, a group of parasitic protozoa. The RNA editing process in trypanosomes is called kinetoplastid RNA (kRNA) editing, after the kinetoplastid containing mitochondria, and creates translatable open reading frames by inserting and/or deleting uridine bases within the encoded mRNA sequence. The main proteins responsible for the kRNA editing process have been characterized and form a 20S editosome. Other accessory proteins have also been implicated in the editing process. Recently, the multiprotein mitochondrial RNA binding complex 1 (MRB1) has emerged as a key player in this process. One key component predicted to be involved in the MRB1 complex is MRB1590. In vivo experiments indicate a role for MRB1590 in editing mitochondrial mRNA transcripts, in particular the transcript encoding the ATP synthase subunit 6 (A6). The goals of this work were to structurally and biochemically characterize MRB1590 to characterize its role in the kRNA editing process, specifically in the editing of the A6 transcript.

The structure of MRB1590 was solved using X-ray crystallography under three different conditions. The overall structure shows the protein is dimeric and contains a central ABC-ATPase fold embedded between a novel N- and C-terminal domain, with a pore being created between the N-terminal domains. The structure of MRB1590 was solved in the presence of ADP and AMP-PNP, showing the nucleotide bound in a pocket located in the ABC-ATPase fold, similar to other ABC-ATPase proteins. The structure of MRB1590 solved in the presence of ADP and RNA revealed a distinct conformation compared to the other two structures in which it adopted an “open” state with a significantly expanded pore. These combined structures suggest that MRB1590 is in equilibrium between open and closed states with ADP binding favoring the open conformation allowing RNA to bind in the pore.

Fluorescence polarization (FP) experiments showed that MRB1590 binds with significantly enhanced affinity to a GC-rich RNA sequence from the A6 transcript compared to other RNA sequences that were tested. Because the MRB1590 structures predict the RNA binding pocket to be the pore created by N-terminal domains, basic residues present in this region were mutated and the effect on RNA binding was tested. The RNA binding of MRB1590 pore mutants was reduced, indicating that these residues are involved in RNA binding. Our findings support MRB1590 as a kRNA editing accessory protein and suggest it may act as an RNA chaperone that ensures the complete editing of the A6 transcript.