RNA editing catalytic complexes edit multiple mRNA sites non-processively in Trypanosoma brucei

RNA editing generates mature mitochondrial mRNAs in T. brucei by extensive uridine insertion and deletion at numerous editing sites (ESs) as specified by guide RNAs (gRNAs). The editing is performed by three RNA Editing Catalytic Complexes (RECCs) which each have a different endonuclease in addition to 12 proteins in common resulting in RECC1 that is specific for deletion ESs and RECC2 and RECC3 that are specific for insertion ESs. Thus, different RECCs are required for editing of mRNA sequence regions where single gRNAs specify a combination of insertion and deletion ESs. We investigated how the three different RECCs might edit combinations of insertion and deletion ESs that are specified by single gRNAs by testing whether their endonuclease compositions are stable or dynamic during editing. We analyzed in vivo BirA* proximity labeling and found that the endonucleases remain associated with their set of common RECC proteins during editing when expressed at normal physiological levels. We also found that overexpression of endonuclease components resulted in minor effects on RECCs but did not affect growth. Thus, the protein stoichiometries that exist within each RECC can be altered by perturbations of RECC expression levels. These results indicate that editing of consecutive insertion and deletion ESs occurs by successive engagement and disengagement of RECCs, i.e., is non-processive, which is likely the case for consecutive pairs of insertion or deletion ESs. This clarifies the nature of the complex patterns of partially edited mRNAs that occur in vivo.

Automated summary

RNA editing in Trypanosoma brucei generates mature mitochondrial mRNAs through extensive uridine insertion and deletion at specific editing sites (ESs) guided by RNA molecules (gRNAs). This process involves three different RNA Editing Catalytic Complexes (RECCs) that each have a unique endonuclease and share 12 common proteins. The study shows that the endonucleases remain associated with their common RECC proteins during editing, and the stoichiometry of the RECCs can be altered by changes in expression levels. The findings suggest that consecutive insertion and deletion ESs are edited non-processively by successive engagement and disengagement of the RECCs.