IDENTIFICATION BY UV CROSS-LINKING OF OLIGO(U)-BINDING PROTEINS IN MITOCHONDRIA OF THE INSECT TRYPANOSOMATID CRITHIDIA-FASCICULATA

RNA editing in trypanosomes is the process of insertion and deletion o f U residues at specific sites of mitochondrial transcripts mediated b y short guide RNAs (gRNAs) that have a 3' oligo(U) extension. Here we describe the identification by UV cross-linking of proteins present in mitochondrial extracts from Crithidia fasciculata with a high affinit y for gRNAs, and the characterization of the binding specificity. A 65 -kDa protein binds to gRNAs provided they are equipped with a U tail, to post-transcriptionally labelled mitoribosomal 9S and 12S RNAs that also possess a 3' terminal stretch of U residues, and to free oligo(U) sequences with a minimal length of 23-29 nucleotides. It does not bin d to a number of control RNAs, one of which has an internal U stretch of 13 residues. Poly(U), but not poly(C) or total yeast RNA, efficient ly competes for binding to gRNA. Proteins of 88 kDa and 30 kDa also bi nd to gRNAs with a U tail, to miochondrial ribosomal RNAs and to oligo (U). These proteins, however, require longer oligo(i) for binding (>39 nucleotides) and they also have an affinity for other U-rich RNAs and poly(C). For comparison, part of the analysis was also carried out wi th a mitochondrial extract from Trypanosoma brucei. in this organism, gRNA-binding proteins of 83 kDa and 63 kDa were found with the same pr eference for 3'-terminal oligomeric U stretches as the C. fasciculata 65-kDa protein, whereas the binding specificity of a 26-kDa protein re sembled that of the C. fasciculata 88-kDa and 30-kDa proteins. The pos sible involvement of the proteins in the editing process is discussed.