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.

The basic mechanism of uridine insertion/deletion RNA editing in mitochondria of kinetoplastid protists has been established for some time but the molecular details remained largely unknown. Recently, there has been significant progress in defining the molecular components of the editing reaction. A number of factors have been isolated from trypanosome mitochondria, some of which have been definitely implicated in the uridine insertion/deletion RNA editing reaction and others of which have been circumstantially implicated. Several protein complexes have been isolated which exhibit some editing activities, and the macromolecular organization of these complexes is being analyzed. In addition, there have been several important technical advances in the in vitro analysis of editing. In this review we critically examine the various factors and complexes proposed to be involved in RNA editing.

We characterized the genes coding for the two dedicated enzymes of ethanolic fermentation, alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC), and show that they are functional in pollen. Two PDC-encoding genes were isolated, which displayed reciprocal regulation: PDC1 was anaerobically induced in leaves, whereas PDC2 mRNA was absent in leaves, but constitutively present in pollen. A flux through the ethanolic fermentation pathway could be measured in pollen under all tested environmental and developmental conditions. Surprisingly, the major factor influencing the rate of ethanol production was not oxygen availability, but the composition of the incubation medium. Under optimal conditions for pollen tube growth, approximately two-thirds of the carbon consumed was fermented, and ethanol accumulated into the surrounding medium to a concentration exceeding 100 mM.

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We describe here the isolation and characterization of a novel RNA-binding protein, RBP38, from Leishmania tarentolae mitochondria. This protein does not contain any known RNA-binding motifs and is highly conserved among the trypanosomatids, but no homologues were found in other organisms. Recombinant LtRBP38 binds single and double-stranded (ds) RNA substrates with dissociation constants in the 100 nM range, as determined by fluorescence polarization analysis. Downregulation of expression of the homologous gene, TbRBP38, in procyclic Trypanosoma brucei by using conditional dsRNA interference resulted in 80% reduction of steady-state levels of RNAs transcribed from both maxicircle and minicircle DNA. In organello pulse-chase labeling experiments were used to determine the stability of RNAs in mitochondria that were depleted of TbRBP38. The half-life of metabolically labeled RNA decreased from ϳ160 to ϳ60 min after depletion. In contrast, there was no change in transcriptional activity. These observations suggest a role of RBP38 in stabilizing mitochondrial RNA.RNA-binding proteins are involved in the synthesis, processing, transport, translation, degradation, and stabilization of RNA (7, 24). The mitochondrial genome of trypanosomatids is composed of thousands of circular DNA molecules, which are catenated and form a dense body of DNA, called the kinetoplast DNA network. This consists of two types of molecules: maxicircles and minicircles. The maxicircle encodes two rRNAs, 18 mRNAs, and a few gRNAs (2). The minicircle encodes the majority of the gRNAs (28). The maxicircle protein coding regions are very compact, and many of the genes overlap extensively, leaving little room for regulatory sequences that might control transcription and RNA processing. It was shown previously that maxicircle transcription produces polycistronic RNAs that are processed to mature RNAs (16,27). The presence of such precursors indicates that the mature RNAs are generated by 3Ј-and 5Ј-end processing, which, for several of the overlapping genes, eliminates a portion of the coding regions of the adjacent transcripts, which then must be degraded. Mitochondrial biogenesis in Trypanosoma brucei is developmentally regulated (23,26,29). In long slender bloodstream forms mitochondrial function is strongly repressed.During differentiation, the mitochondrion is partially activated in the short stumpy bloodstream form, but only in the insect midgut procyclic form is the mitochondrion fully active. This change in physiology is reflected in a stage-specific regulation of mitochondrial gene expression (15, 18). The levels of some mitochondrial transcripts in short stumpy cells are intermediate between the long slender and procyclic forms, whereas other transcripts are already at the procyclic levels. The transcription rates and processing of the rRNA genes are the same in the bloodstream and the procyclic forms, suggesting that the difference in abundance of the mature rRNAs is due to differential stability in the two forms of the life cycle (19). T...

In this paper we report the first case of antimycin A resistance in a protozoan parasite that is attributable to a mutation in the mitochondrial apocytochrome b (CYb) gene. We selected for, and isolated, a mutant Leishmania tarentolae strain that is resistant to antimycin A. This resistance was evident at the levels of the in vitro growth and enzymatic activity of the cytochrome bc1 complex. Molecular characterisation of the mutant revealed a Ser35Ile mutation in the expected region of the CYb gene. In kinetoplastids, CYb and other structural genes of the mitochondrial genome are located on the maxicircle component of the mitochondrial DNA, which is present in 20-50 copies. Primer-extension analysis confirmed the presence of the mutation at the mRNA level. The phenotypic manifestation of the mutation implies that the CYb mRNA is edited and translated within the mitochondrion. Thus, this finding provides direct evidence that edited RNAs are translated in kinetoplastid mitochondria. Furthermore, a defined mutation conferring drug resistance to a mitochondrial gene product can be exploited for the development of mitochondrial transfection systems for trypanosomatids.

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