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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 ...

A 3' terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei. The enzyme is specific for 3' U-addition in the presence of Mg(2+). TUTase is present in vivo in at least two stable configurations: one contains a approximately 500 kDa TUTase oligomer and the other a approximately 700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect parasite viability.

Amplification of DNA sequences from the kinetoplast minicircle DNA was employed as a method for the detection and classification of small numbers of Trypanosoma cruzi cells. Two overlapping fragments from the conserved 120 bp minirepeat regions of the minicircle DNA and one fragment covering the adjacent variable regions were amplified. The minimal amount of minicircle DNA required to detect a product by hybridization with an oligonucleotide probe was 0.015 fg, which represents approximately 10 molecules or 0.1% of the minicircle DNA component of a single cell. The amplification worked equally well with kDNA from several strains of T. cruzi and did not occur with kDNA from several other kinetoplastids. kDNA recovered from less than 10 trypanosomes in whole blood could be used as a template for amplification; the presence of a several billion fold excess of human DNA had no effect on the amplification process. Schizodeme analysis by hybridization with specific oligonucleotides or by direct restriction enzyme digestion could be performed on the amplified fragments representing the minicircle conserved region or variable regions. This method should prove useful as a rapid, specific and sensitive assay for Chagas' disease in chronic patients as well as for epidemiological studies of infected animals and insects.

The insertion and deletion of U residues at specific sites in mRNAs in trypanosome mitochondria is thought to involve 3 terminal uridylyl transferase (TUTase) activity. TUTase activity is also required to create the nonencoded 3 oligo[U] tails of the transacting guide RNAs (gRNAs). We have described two TUTases, RET1 (RNA editing TUTase 1) and RET2 (RNA editing TUTase 2) as components of different editing complexes. Tandem affinity purificationtagged Trypanosoma brucei RET2 (TbRET2) was expressed and localized to the cytosol in Leishmania tarentolae cells by removing the mitochondrial signal sequence. Double-affinity isolation yielded tagged TbRET2, together with a few additional proteins. This material exhibits a U-specific transferase activity in which a single U is added to the 3 end of a single-stranded RNA, thereby confirming that RET2 is a 3 TUTase. We also found that RNA interference of RET2 expression in T. brucei inhibits in vitro Uinsertion editing and has no effect on the length of the 3 oligo[U] tails of the gRNAs, whereas down-regulation of RET1 has a minor effect on in vitro U-insertion editing, but produces a decrease in the average length of the oligo[U] tails. This finding suggests that RET2 is responsible for U-insertions at editing sites and RET1 is involved in gRNA 3 end maturation, which is essential for creating functional gRNAs. From these results we have functionally relabeled the previously described TUT-II complex containing RET1 as the guide RNA processing complex.U -insertion͞deletion RNA editing in trypanosome mitochondria involves the annealing of a guide RNA (gRNA) to an mRNA, endonuclease cleavage at a non-base-paired editing site adjacent to the RNA duplex, addition or deletion of Us to or from the 3Ј end of the 5Ј cleavage fragment, and religation of the two mRNA fragments (1-4). Each gRNA mediates the editing of 1-5 sites in a 3Ј to 5Ј polarity, and multiple overlapping gRNAs mediate the editing of an entire domain, also in a 3Ј to 5Ј polarity (5).We previously isolated the RNA editing terminal uridylyl transferase (TUTase) 1 (RET1) and showed it to be present both as a free tetramer and as a component of a high molecular weight complex (TUT II) (6). This complex migrates on a native gel at Ϸ700 kDa and interacts in an RNase-sensitive manner with the larger ligase-containing complex (L-complex) (7). Downregulation of RET1 expression in Trypanosoma brucei by conditional RNA interference (RNAi) caused a decrease in the steady-state abundance of edited mRNAs without any effect on transcription (6). The core L-complex and associated proteins, capable of both U-insertion and U-deletion editing activity in vitro, was isolated from Leishmania tarentolae mitochondrial extract by tandem affinity purification (TAP) (8), followed by glycerol gradient sedimentation (7). The L-complex contains two adenylatable RNA ligases, REL1 and REL2, three related zinc finger-containing proteins, two proteins with RNase III motifs, two proteins with an AP endonuclease exonuclease phosphatase motif, a se...

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Seventeen kinetoplast minicircle-encoded and nine maxicircle-encoded gRNA genes have been identified. Six overlapping minicircle-encoded gRNAs mediate editing for the 5'-pan-edited MURF4 gene and two for the 5'-edited COIII gene. The pan-edited RPS12 mRNA is edited by seven minicircle-encoded gRNAs and one maxicircle-encoded gRNA. The 3'-most gRNA in each domain forms an anchor with unedited mRNA, whereas upstream gRNAs form anchors only with edited mRNA, thereby explaining the observed 3' to 5' polarity of editing within an editing domain. We suggest that a role of G-U base pairs is to allow breathing of the edited mRNA-gRNA hybrid and formation of the upstream anchor hybrid.