Quantitation of RNA editing substrates, products and potential intermediates: implications for developmental regulation.

Abstract

Kinetoplast mitochondrial RNA editing is the developmentally regulated post-transcriptional process of uridine insertion and deletion in mRNAs directed by short guide RNAs (gRNAs), which creates functional mRNAs. Two mechanisms are proposed: transesterification which predicts gRNA/mRNA chimeric intermediates, and enzymatic steps which allow but do not require chimeric intermediates. We quantitated the copy number of apocytochrome b (CYb) gRNAs, edited/unedited mRNAs and gRNA/mRNA chimeras in bloodstream and procyclic form cells of Trypanosoma brucei. Both forms have 35 copies/cell of two gRNAs. Bloodstream forms contain 15 unedited and edited CYb mRNA molecules/cell while procyclic forms have four times as much unedited and over 10 times as much edited mRNA. Chimera levels are very low, 350-5000-fold lower than unedited mRNA or gRNAs, but are over 10 times more abundant in procyclic than bloodstream forms. These results are consistent with chimeras being editing intermediates if their resolution is rapid in respect to their formation, although they could be non-productive byproducts of the editing reaction. Bloodstream chimera sequences differ from procyclic chimeras. These results indicate that developmental regulation is not by gRNA abundance and suggest that it occurs at the level of gRNA utilization possibly by changing abundance of unedited CYb mRNA.