QUANTITATION OF RNA EDITING SUBSTRATES, PRODUCTS AND POTENTIAL INTERMEDIATES - IMPLICATIONS FOR DEVELOPMENTAL REGULATION

Kinetoplast mitochondrial RNA editing is the developmentally regulated post-transcriptional process of uridine insertion and deletion in mRN As directed by short guide RNAs (gRNAs), which creates functional mRNA s, Two mechanisms are proposed: transesterification which predicts gRN A/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 chime ras in bloodstream and procyclic form cells of Trypanosoma brucei. Bot h forms have 35 copies/cell of two gRNAs, Bloodstream forms contain 15 unedited and edited CYb mRNA molecules/cell while procyclic forms hav e four times as much unedited and over 10 times as much edited mRNA. C himera levels are very low, 350-5000-fold lower than unedited mRNA or gRNAs, but are over 10 times more abundant in procyclic than bloodstre am forms, These results are consistent with chimeras being editing int ermediates if their resolution is rapid in respect to their formation, although they could be non-productive byproducts of the editing react ion. Bloodstream chimera sequences differ from procyclic chimeras, The se results indicate that developmental regulation is not by gRNA abund ance and suggest that it occurs at the level of gRNA utilization possi bly by changing abundance of unedited CYb mRNA.