A multiprotein, high molecular weight complex active in both U-insertion and U-deletion as judged by a pre-cleaved RNA editing assay was isolated from mitochondrial extracts of Leishmania tarentolae by the tandem af®nity puri®cation (TAP) procedure, using three different TAP-tagged proteins of the complex. This editing-or E-complex consists of at least three protein-containing components interacting via RNA: the RNA ligase-containing L-complex, a 3¢ TUTase (terminal uridylyltransferase) and two RNA-binding proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components were identi®ed by mass spectrometric analysis of the core L-complex: two RNA ligases; homologs of the four Trypanosoma brucei editing proteins; and seven novel polypeptides, among which were two with RNase III, one with an AP endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins have no similarities beyond kinetoplastids. Keywords: editosome/RNA editing/TAP/TUTase Introduction Uridine insertion/deletion RNA editing is a post-transcriptional RNA modi®cation phenomenon that occurs in the mitochondrion of kinetoplastid protists . The mechanism involves the initial hybridization to an mRNA of a complementary guide RNA (gRNA) which guides a speci®c endonuclease cleavage at the ®rst editing site . This is followed by either deletion of the unpaired uridines from the cleavage fragment or the 3¢ addition to the mRNA 5¢ cleavage fragment, hybridization of the added Us to the guiding nucleotides in the gRNA, and religation of the two mRNA cleavage fragments. Each gRNA speci®es the 3¢ to 5¢ editing of a small number of sites and, in the case of a multiple gRNA-mediated editing domain, creates the anchor sequence for hybridization of the adjacent upstream gRNA, thus producing an overall 3¢ to 5¢ progression of editing. A minimal non-progressive editing activity at one or two sites has been demonstrated in vitro using crude or partially puri®ed mitochondrial extract, and the reaction was shown to involve high molecular weight RNP complexes (Byrne et al., 1996;Cruz-Reyes and Sollner-Webb, 1996;Kable et al., 1996;Seiwert et al., 1996). The mechanism described above was proposed >12 years ago , and was veri®ed experimentally in 1996 for both Trypanosoma brucei and Leishmania tarentolae (Byrne et al., 1996;Cruz-Reyes and Sollner-Webb, 1996;Seiwert et al., 1996). However, progress in the identi®cation of speci®c proteins involved in editing has been hampered by their low abundance and by the low ef®ciency of the in vitro editing assays. A seven polypeptide complex from T.brucei mitochondria that supported in vitro insertion and deletion editing was isolated by two chromatographic steps and was proposed to represent a core editing complex (Rusche et al., 1997). An~20 polypeptide complex with similar activities was isolated in another laboratory by a similar fractionation (Panigrahi et al., 2001a,b).The genes for several of the major components of these complexes have been identi®ed, but only a few proteins so far have been ascribed ...
