The overall goal of the research described in this grant application is to better understand a very bizarre and novel process of transcript maturation that involves the often very extensive insertion (and occasional deletion) of U residues from primary transcripts to achieve the mature RNA sequences. This """"""""RNA editing"""""""" has thus far been shown to be utilized by mitochondrial RNAs of trypanosomes but it may well be used by other organisms as well and is thought to provide clues to the early evolution of RNA metabolism This U insertion/deletion is """"""""guided"""""""" by short complementary RNAs (gRNAs) which evidently also serve to donate their U residues to the mRNA through formation of a chimeric intermediate with the mRNA portion downstream of the site of editing. But how these gRNAs do the guiding and what is the mechanism of formation of the chimeric intermediate and of the entire reaction are some of the crucial, unresolved questions about RNA editing that we plan to address. Using a trypanosome mitochondrial extract recently shown to form such gRNA/mRNA chimeras in vitro, we will investigate the mechanism/catalytic basis of this chimera formation. To help determine whether the in vitro chimera formation occurs by transesterification or by an endonuclease/ligase route, we will identify the precise site of the junction and the stereochemical considerations imposed on the junction phosphate of the chimer~ We will further analyze chimera formation by studying known co- fractionating activities and by further purification of the activity. We will also attempt to recreate an entire editing reaction in vitro (preliminary very encouraging results have now shown specific cleavage of the gRNA/mRNA chimera and reformation of an intact partly edited mRNA!) and then will study this reaction. Analysis in permeabiIized cells should provide much needed information about the kinetics of the editing process, including the production of the surprisingly abundant aberrant edited molecules. In complementary studies of the steady state mitochondrial RNA population, we will clone and analyze various intermediates in the editing process, asking whether these molecules correspond to predictions from transesterification or nuclease/ligase models of editing, whether editing occurs in strict 3'-> 5' order or shows some flexibility, and whether activities suggested from the in vitro analysis are utilized for editing in vivo. We will use in situ hybridization analysis to localize where within the trypanosome mitochondria the unedited, the partially edited, and possibly the fully edited RNAs are located. This information could allow determination of whether editing occurs in the kinetoplast mitochondrial body or in the long mitochondrial tubule that extends from it along the length of the trypanosome. Through these studies we hope to gain a better understanding about this very ancient and novel form of RNA maturation.
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