Research in the Simpson Laboratory

The trypanosomes or kinetoplastid protozoa comprise a large group of parasitic flagellated cells that are the causal agents for a variety of human and animal diseases. Research in Dr. Simpson's laboratory has focused on the molecular biology of the mitochondrial genome in these organisms from several points of view. These cells represent one of the earliest eukaryotic lineages containing mitochondria and, as such, possess many unusual physical and biochemical features, among which is a mitochondrial genome known as 'kinetoplast DNA' that consists of a network of thousands of catenated mini- and maxicircles, transcripts of which are modified by a novel process termed 'RNA editing'. Click here to see several micrographs of kDNA networks.

The cells can be plated on agar. Click here to see a movie of T. brucei procyclic colonies on agar.

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RNA Editing of Mitochondrial Transcripts in the Mitochondrion of Trypanosomatids

RNA editing in trypanosomatid protozoa involves the insertion and deletion of uridine residues (U's) at specific sites within coding regions of mRNA transcripts of the maxicircle genome. The sequence information for editing is contained in a class of small RNAs termed guide RNAs(gRNAs), which were previously discovered in this laboratory. gRNAs are small RNAs which, at the 5' end have a region of complementarity with mRNA sequence just downstream of the sites to be edited, and at the 3' end have a non-encoded oligo[U] tail. Click here to see several examples of edited sequence aligned with the cognate gRNAs.

The majority of the gRNAs are encoded in the thousands of minicircle molecules which are catenated together into a single giant network of DNA. This laboratory showed previously that the 3' to 5' polarity of editing is due to the creation of upstream gRNA anchor sequences by downstream editing. Here is a figure of the pan-edited RPS12 gene

Two basic models have been proposed by our laboratory for the mechanism of editing, one of which involves a cleavage, 3'-terminal U addition, and religation.

The other, which was proposed independently by Cech, involves two successive transesterifications such as occur in RNA splicing, with a transfer of U's from the 3' end of the gRNA or directly from UTP to the editing site. gRNA/mRNA chimeric molecules, which were discovered by the Simpson laboratory, were initially proposed to be intermediates in the transesterification model, but recent evidence suggests that these molecules represent non-productive byproducts of the cleavage-ligation process.

The Evolution of RNA Editing

A rooted phylogenetic tree of the kinetoplastid protozoa was constructed from nuclear rRNA sequences, which, together with a comparative analysis of editing of three maxicircle genes in several trypanosomatid species, led to the surprising conclusion that extensive or pan-editing, mediated by multiple overlapping guide RNAs, is phylogenetically more ancient than the limited editing which occurs at the 5' end of editing domains (5'-editing), and that RNA editing preceded the evolution of parasitism in this group.

The maxicircle genomic organization in all analyzed trypanosomatid species is identical, but the extent of editing varies considerably (Click here for NAR cover showing a comparison of maxicircle genomes). This can be visualized in a comparison of the extent of editing of the A6 or MURF4 mRNA in three species. The editing appears to become limited to the 5' end of the editing domain. This can be also seen in a comparison of the editing of the ND7 mRNA in T. brucei and L. tarentolae. In T. brucei there is pan-editing of two domains, whereas in L. tarentolae, editing is limited to the 5' end of each domain.

The phylogenetic analysis also showed that the bodonid/cryptobiids represent an early diverged sister group to the trypanosomatids, as was proposed previously by classical analyses. Click here to see a diagram of the taxonomy of kinetoplastid protozoa. Analysis of one cryptobiid species, Trypanoplasma borreli, which is discussed below, showed the presence of the U addition/deletion type of RNA editing of several mitochondrial transcripts, in spite of a complete dissimilarity of the mitochondrial gene order.

The evidence indicates that ancestral kinetoplastid cryptogenes were probably pan-edited and the 5'-edited homologues were possibly generated by several independent retroposition events from partially edited RNAs. A comparison of the extent of editing in an old laboratory strain of Leishmania tarentolae and a recently isolated strain has provided additional evidence for this mechanism.

This research was done by Dimitri Maslov and Otavio Thiemann.

Disruption of RNA Editing by Prolonged Culture of the Promastigote Form

RNA editing in kinetoplastids appears to be a labile genetic trait that is affected by prolonged cell culture. The transcripts of the G1-G5 cryptogenes are pan-edited in the recently isolated LEM125 strain of Leishmania tarentolae, but not in the UC strain which has been in culture for 55 years. At least 32 minicircle-encoded guide RNAs encoding gRNAs for the editing of these transcripts were lost during the culture history of the old lab strain, probably due to the absence of a selective pressure for the protein products, which include subunits of complex I of the respiratory chain. See also guide RNAs2.

Click here to see a diagram of the construction of the gRNA library which was used to detect the additional gRNAs in the LEM125 strain..

The absence of gRNAs for the editing of G5 in the UC strain led to the existence of extensively misedited RNAs. Some of these misedited RNAs showed correct editing of block I, and misediting upstream. The editing of Block I was mediated by a maxicircle-encoded gRNA, which was present in both strains. Several non-cognate gRNAs were identified in the UC strain which could account for specific misedited upstream sequences.

This research was done by Otavio Thiemann.

Diagnosis of Chagas Disease

Trypanosoma cruzi is the causal agent of Chagas Disease, an important disease of the American tropics, for which there is no adequate chemotherapy or vaccine. A knowledge of the RNA editing system and the genetic function of these molecules could aid in development of a rational chemotherapy for this disease. The previously observed extensive sequence heterogeneity of the kinetoplast minicircle DNA in Trypanosoma cruzi, both intra- and inter-strain, has raised the question as to how the minicircle DNA in this species can have any gRNA-coding capacity at all, since there does not appear to be any variable region sequences conserved between different strains. To address this question, the complete edited sequence of maxicircle unidentified reading frame 4 mRNA was obtained and we were able to identify 25 cognate gRNAs from gRNA libraries constructed from two clonal strains of T. cruzi - Sylvio X10/CL1 and CAN III/CL1.

 Libraries of PCR-amplified minicircle variable regions were also constructed for both strains. A single gene for each gRNA was identified in the same polarity within specific minicircle variable regions from both strains, 60-100 nucleotides downstream from the conserved 12mer sequence. GTP-capped total gRNA from one strain failed to cross-hybridize with minicircle DNA from the other strain. The explanation for this proved to be the number of polymorphisms, mainly transitions, within the homologous gRNAs in the two strains. In most cases these transitions did not destroy the edited mRNA/gRNA base-pairing, as a result of the allowed G-U wobble base-pairing. The sequences of the variable regions containing homologous gRNAs in the two strains probably derived from an ancestral sequence, and each has accumulated sufficient polymorphisms so as not to allow hybridization. Within a strain, multiple redundant gRNAs were identified which encode identical editing information but have different sequences.

 Dr. Simpson's laboratory previously developed a sensitive diagnostic test for the presence of these parasites in chronically ill patients by PCR amplification of parasite-specific but highly polymorphic DNA fragments from kinetoplast minicircle molecules. A single pair of PCR primers within a conserved region of the mini-exon repeat was used to amplify the repeats from 10 species of pathogenic Leishmania belonging to 4 major clinical groups and also from 3 species of Trypanosoma.

Oligonucleotide hybridization probes for the detection and identification of the PCR-amplified mini-exon repeats were constructed from alignments of mini-exon intron and intergenic sequences. The probes generated from mini-exon intergenic regions of the L. (V.) braziliensis, L. (L.) donovani and L. (L.) mexicana species hybridized specifically to their cognate groups without discriminating between the species within the groups. The probes for L. (L.) major and L. (L.) aethiopica were species-specific, while the L. (L.) tropica probe also hybridized with the L. (L.) aethiopica mini-exon repeat. The mini-exon intron-derived probes for T. cruzi, T. rangeli and T. brucei were species-specific. This method involving the detection of specific PCR-amplified products produced using a single primer set represents a novel sensitive and specific assay for multiple trypanosomatid species and groups. It essentially represents a multiplex assay and should complement the minicircle-based diagnostic assay previously developed by this laboratory.

Here is an article on the history of the development of this assay.

This research was done by Herbert Avila et al.

RNA Editing in Crithidia fasciculata

Although the mitochondrial uridine insertion/deletion, guide RNA-mediated type of RNA editing has been described in Crithidia fasciculata, no evidence for the encoding of guide RNAs in the kinetoplast minicircle DNA has been presented. There has also been a question as to the capacity of the minicircle DNA in this species to encode the required variety of gRNAs, since the kinetoplast DNA from the C1 strain has been reported as essentially containing a single minicircle sequence class. To address this problem, the genomic and mature edited sequences of the MURF4 and RPS12 cryptogenes were determined, and a gRNA library was constructed from mitochondrial RNA. Five specific gRNAs were identified, two of which edit blocks within the MURF4 mRNA, and three of which edit blocks within the RPS12 mRNA. The genes for these gRNAs are all localized with identical polarity within one of the two variable regions of specific minicircle molecules, approximately 60 bp from the 'bend' region. These minicircles were found to represent minor sequence classes representing approximately 2% of the minicircle DNA population in the network. The major minicircle sequence class also encodes a gRNA at the same relative genomic location, but the editing role of this gRNA was not determined. These results confirm that kinetoplast minicircle DNA molecules in this species encode gRNAs, as is the case in other trypanosomatids, and suggest that the copy number of specific minicircle sequence classes can vary dramatically without an overall effect on the RNA editing system.

This research was done by Shinji Yasuhira.

Guide RNAs and guide RNA genes in the cryptobiid kinetoplastid protozoan, Trypanoplasma borreli

Trypanoplasma borreli belongs to the bodonid/cryptobiid group of kinetoplastid protozoa, which represents a sister group to the trypanosomatids. RNA transcripts from several mitochondrial genes in this organism undergo the trypanosomatid type of uridine addition/deletion RNA editing. A gRNA cDNA library was constructed and five gRNAs were identified, one for editing the ribosomal protein S12 mRNA, three for editing the cytochrome oxidase subunit I mRNA and one for editing the cytochrome b mRNA. All of the gRNAs contained non-encoded oligo[U] sequences at their 5' end, as well as at the 3' end as is common with gRNAs in trypanosomatids. The mechanism for addition of the 5' non-encoded oligo[U] sequence and the function of this sequence are unknown. The T. borreli gRNAs were shorter (25-35 nt, excluding the 5' oligo[U]) than gRNAs in trypanosomatids (45-50 nucleotides), indicating a smaller size of editing blocks in this organism. Genomic sequences for two gRNAs were cloned and sequenced. These two gRNA-encoding sequences were shown to originate from the 170-200 kb Component I molecules which represent a possible homologue of minicircle DNA in trypanosomatids, and not from the 80 kb Component II molecules which contain the structural genes and cryptogenes.

Comparative genomic organization of gRNAs in minicircles from several species

This research was done by Shinji Yasuhira

Phylogenetic affinity of mitochondria of Euglena gracilis and kinetoplastids using cytochrome oxidase I and hsp60.

The mitochondrial DNA-encoded cytochrome oxidase subunit I (COI) gene and the nuclear DNA-encoded hsp60 gene from the euglenoid protozoan Euglena gracilis were cloned and sequenced. The COI sequence represents the first example of a mitochondrial genome-encoded gene from this organism. This gene contains seven TGG tryptophan codons and no TGA tryptophan codons, suggesting the use of the universal genetic code. This differs from the situation in the mitochondrion of the related kinetoplastid protozoa, in which TGA codes for tryptophan. In addition, a complete absence of CGN triplets may imply the lack of the corresponding tRNA species. COI cDNAs from E. gracilis possess short 5' and 3' untranslated transcribed sequences and lack a 3' poly[A] tail. The COI gene does not require uridine insertion/ deletion RNA editing, as occurs in kinetoplastid mitochondria, to be functional, and no short guide RNA-like molecules could be visualized by labeling total mitochondrial RNA with [alpha-32P]GTP and guanylyl transferase. In spite of the differences in codon usage and the 3' end structures of mRNAs, phylogenetic analysis using the COI and hsp60 protein sequences suggests a monophyletic relationship between the mitochondrial genomes of E. gracilis and of the kinetoplastids, which is consistent with the phylogenetic relationship of these groups previously obtained using nuclear ribosomal RNA sequences.

This research was done by Shinji Yasuhira.

Involvement of mitochondrial ribonucleoprotein complexes in RNA Editing

By analysis of mitochondrial extracts on glycerol gradients, two types of ribonucleoprotein (RNP) complexes were identified containing gRNAs. The T-class sediments at approximately 10S and consists of approximately six complexes, the endogenous RNA of which can be self-labeled with [32P]UTP. The most abundant T-complex, T-IV, was visualized by electron microscopy as 980-140 A particles. This complex exhibits terminal uridylyl transferase (TUTase) activity and contains gRNAs. The other T-complexes also contain mRNA fragments. The function of T-IV may be to add U's to the 3' end of the gRNA. The second class of RNP complexes consists of 170-300 A particles which show little TUTase activity, and exhibit an in vitro RNA editing-like activity.

Two mitochondrial proteins of 18 kD and 51 kD which appeared to show interaction with exogenous gRNAs were isolated and the genes cloned. These proteins were components of complex T-I and T-VI, respectively. The proteins possess 17 and 9 amino acid N terminal cleavable mitochondrial targeting sequences. The p18 protein localized throughout the entire tubular mitochondrion. and has been identified by the Benne lab as a subunit of the mtochondrial ATPase. The p51 protein was identified as a mitochondrial aldehyde dehydrogenase. In addition, 71 kD and 62 kD proteins which comigrated in native gels with other T-complexes were identified as hsp70 and hsp60 homologs.

The majority of the protein-RNA interactions in the T-complexes were shown to be low affinity. However, digestion of the extract with micrococcal nuclease and saturation with rRNA was found to uncover a high affinity RNP complex, which involves at least two proteins interacting with the endogenous gRNAs.

The second class of complexes sediments at 20S. A gRNA-independent in vitro editing-like activity which comigrates with the 20S complexes was assayed by following the incorporation of radioactively labeled U's into the pre-edited region of a synthetic mRNA substrate by digestion with RNase H and specific oligonucleotides. The incorporation is limited precisely to the pre-edited region and is dependent on some type of endogenous RNA, as shown by micrococcal nuclease digestion experiments. Labeled C residues were incorporated into the same sites as U residues, but at a 20 fold lower level.

This research was done by Marian Peris, Agda Simpson, Frederic Bringaud, Elaine Byrne and Georges Frech.

Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract

Two polypeptides of 50 and 45 kDa were adenylated by incubation of a mitochondrial extract from Leishmania tarentolae with [a-32P]ATP. These proteins were components of the 20S complex, which migrated as a single band of approximately 1800 kDa in a native gel. The facts that RNA ligase activity cosedimented at 20S and that the ATP-labeled p45 and p50 polypeptides were deadenylated upon incubation with a ligatable RNA substrate suggests that these proteins may represent charged intermediates of a mitochondrial RNA ligase. Hybridization of native gel blots with guide RNA (gRNA) probes showed the presence of gRNA in the previously identified T-IV complexes that sedimented in glycerol at 10S and contained TUTase activity, and also in a previously unidentified class of heterodisperse complexes that sedimented throughout the gradient. gRNAs were not detected in the p45+p50-containing complex. The heterodisperse gRNA-containing complexes were sensitive to incubation at 27oC and appear to represent complexes of T-IV subunits with mRNA. Polyclonal antiserum to a 70 kDa protein that purified with terminal uridylyl transferase activity was generated, and the antiserum was used to show that this p70 polypeptide was a component of both the T-IV and the heterodisperse gRNA-containing complexes. We propose that the p45+p50-containing 1800 kDa complex and the p70+gRNA -containing heterodisperse complexes interact in the editing process.

This research was performed by Marian Peris, Agda M. Simpson, Jeremy Grunstein, Joanna E. Liliental and Georges C. Frech

Guide RNA-independent RNA editing in vitro

A mitochondrial extract from Leishmania tarentolae directs the incorporation of uridylate (U) residues within the pre-edited domain of synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has several characteristics of an in vitro RNA editing activity, but no direct evidence for involvement of guide RNAs was obtained. In fact, evidence obtained by Greg Connell indicates that this activity is independent of both exogenous and endogenous gRNAs. The incorporation is limited to the preedited region but the number and localization of U's inserted is not identical to that found in mature correctly edited RNA. The activity is selectively inhibited by digestion of the lysate with micrococcal nuclease, possibly suggesting a requirement for some type of endogenous RNA. A low level of incorporation of [alpha-32P]CTP occurs at the same sites as UTP.

Stereochemical evidence for the enzyme cascade model for RNA editing

Chiral phosphorothioates were used to investigate the stereoconfiguration requirements and the stereochemical course of an RNA editing-like internal uridine (U)-incorporation activity and a 3' terminal U-addition activity using a mitochondrial extract from L. tarentolae. The extract utilizes SP-[a-S]UTP for both 3' and internal U- incorporation into substrate RNA. The internal as well as the 3' incorporation of SP-[a-S]UTP proceeds via inversion of the stereoconfiguration. The mitochondrial RNA ligase produces an inversion of the stereoconfiguration. Iternal U-incorporation does not occur at sites containing thiophosphodiesters of the RP configuration. The results are compatible with an enzyme cascade model for this in vitro U-insertion activity involving sequential endonuclease, uridylyl transferase directly from UTP and RNA ligase steps, and incompatible with models involving the transfer of U residues from the 3' end of gRNAs.

This research was done by Georges Frech.

The role of mRNA structure in guide RNA-independent RNA editing in vitro

A primer extension assay was used for the detection of uridine insertions occurring in vitro in synthetic pre-edited cytochrome b mRNA during incubation with a Leishmania tarentolae mitochondrial extract. Two different activities were detected that inserted uridines within the first two editing sites: one that is dependent on the secondary structure of the mRNA but is independent of both exogenous and endogenous guide RNA, and a second that does not put the same structural constraints on the mRNA, but is dependent on the presence of a cognate guide RNA. The possibility that contaminating gRNA-mRNA chimeric molecules were the source of the extension ladders was eliminated by performing the extension assay on RNAs transcribed from 3'-tagged RT-PCR-amplified cDNAs. Leaving aside the question of the biological relevance of the gRNA-independent reaction, it is possible that the structure of the RNAs supporting this in vitro reaction could be mimicking the RNA structures occurring during gRNA-mediated editing. For example, the duplex formed by foldback of the Cyb mRNA 5' and 3' extensions, which is required for the gRNA-independent reaction, may serve the same function as the anchor duplex created by the gRNA binding to the mRNA. Annealing of the cognate gRNA containing a complementary anchor sequence with the 5'-extended mRNA construct would be predicted to disrupt the mRNA secondary structure, thereby accounting for the observed inhibitory effect of the addition of exogenous gRNA on the gRNA-independent U-insertion activity. The creation of a recognition element for the assembly of the U-insertion machinery may represent an important role of the gRNA in the gRNA-mediated reaction. The recognition element could be the double stranded RNA formed by the gRNA-mRNA interaction that may be mimicked by the intramolecular helix necessary for the gRNA-independent reaction.

This work was performed by Greg Connell and Elaine Byrne.

Guide RNA-directed uridine-insertion RNA editing in vitro

Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial messenger RNAs in kinetoplastid protozoa. The U's are proposed to be derived either from UTP by two successive cleavage-ligations or transesterifications, or from the 3' end of the gRNA by the same mechanisms. By use of a sensitive and specific primer extension assay, we have demonstrated guide RNA-dependent U-insertions into a specific editing site of a preedited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U-insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA-mRNA anchor duplex was necessary for activity. UTP and a-b bond hydrolysis of ATP were required, and the activity was inhibited above 50-100 mM KCl. A guide RNA-independent insertion of up to approximately 13 U's occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the guide RNA-independent U-insertion activity and had no effect on the guide RNA-dependent activity. Blocking the 3'OH of the gRNA had little effect on the gRNA-dependent U-insertion activity. The data are consistent with a modified enzyme cascade model in which the U's are derived directly from UTP.

This work was performed by Elaine Byrne and Greg Connell.

Sequence-dependent importation of tRNAs into the mitochondrion of Leishmania tarentolae

Another topic of research in the Simpson lab is the importation of tRNAs into the kinetoplast- mitochondrion of L. tarentolae. Previous research had shown that no mitochondrial tRNAs were encoded in the maxicircle (or minicircle) genome. Therefore the mitochondrial tRNAs had to be derived from the cytosol and be encoded in nuclear genes. To investigate this, an in vivo transfection method was employed. tRNAIle(UAU) was shown previously by Shi et al., 1994 and Lye et al., 1993 to exclusively localized within the mitochondrion and tRNAGln(CUG) exclusively in the cytosol. L. tarentolae cells were transfected with plasmids encoding either tRNAIle or tRNAGln that were tagged with altered sequences in the D loop, permitting discrimination from the endogenous tRNAs. Primer extension analysis was used to show that the plasmid-encoded genes were expressed and that the tagged tRNAs showed a similar intracellular localization as the endogenous tRNAs. Exchange or deletion of the 5'-flanking genomic sequences had no effect on the expression or mitochondrial localization of the tagged tRNAIle or on the expression or cytosolic localization of the tagged tRNAGln, suggesting that the signals for importation are localized within the tRNA itself. Swapping the D loop+stem from the exclusively cytosolic tRNAGln with that from the tRNAIle produced a partial mitochondrial localization of the plasmid-expressed mutated tRNAGln. However, D loop exchange did not eliminate the mitochondrial localization of the plasmid-expressed mutated tRNAIle, suggesting that tertiary structure or additional sequence elements may be involved in the importation signal.

This research was done by Beatriz Lima.

The mitochondrial glutamate dehydrogenase from Leishmania tarentolae is a guide RNA-binding protein

To identify specific proteins interacting with guide RNAs in mitochondrial ribonucleoprotein complexes from Leishmania tarentolae, fractionated and unfractionated mitochondrial extracts were subjected to UV cross-linking with added labeled gRNA and also with [a-32P]UTP-labeled endogenous RNA. An abundant 110-kD protein (p110) localized in the T-V complex, which sediments in glycerol gradients at the leading edge of the 10S terminal uridylyl transferase peak, was found to interact with both types of labeled RNAs. The p110 protein was gel-isolated and subjected to microsequence analysis, and the gene was cloned. The sequence revealed significant similarity with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised against a recombinant fragment of the p110 gene and was used to demonstrate a stable and specific gRNA-binding activity by co-immunoprecipitation and competitive gel-shift analyses. Complex formation was strongly inhibited by competition with poly[U] or by deletion or substitution of the gRNA 3'-oligo[U] tail. Also, addition of a 3' oligo[U] tail to an unrelated transcript was sufficient for p110 binding. Both the gRNA-binding activity of the p110 protein and in vitro gRNA-independent and gRNA-dependent uridine insertion activities in the mitochondrial extract were inhibited by high concentrations of dinucleotides.

This work was performed by Frédéric Bringaud, Renata Stripecke, Georges C. Frech, Stephen Freedland, Christoph W. Turck and Elaine M. Byrne.

Knockout of the glutamate dehydrogenase gene in bloodstream Trypanosoma brucei in culture has no effect on editing of mitochondrial mRNAs

Glutamate dehydrogenase (GDH) was shown previously to bind the 3' oligo[U] tail of the mitochondrial guide RNAs (gRNAs) of Leishmania tarentolae, apparently in the dinucleotide pocket. Bloodstream Trypanosoma brucei cells in culture represent a good system to investigate the genetic effects of knocking out kinetoplastid nuclear genes to test a role in RNA editing, since editing of several mitochondrial genes occurs but is dispensable for viability. Both GDH alleles of bloodstream T. brucei in culture were replaced by drug resistant markers without any effect on viability. The ratios of edited to unedited mRNAs for several cryptogenes were assayed by primer extension analysis. The steady state abundances of these edited RNAs were unaffected by the double knockout. This evidence suggests that GDH may not play a role in the editing reaction in bloodstream trypanosomes in culture, but this conclusion is tentative since there could be redundant genes for any biological function. We employed a double allelic replacement technique to generate a tetracycline inducible conditional expression of an ectopic copy of the deleted gene in bloodstream trypanosomes in culture. We used this strategy for genes encoding mitochondrial proteins which are not required during this stage of the life cycle, but as a general strategy it should be appropriate for generation of conditional null mutants for essential genes as well.

This work was performed by A. Estevez, F. Kierzenbaum, E. Wirtz, F. Bringaud and J. Grunstein.

The mitochondrion in dividing Leishmania tarentolae cells is symmetric and circular and becomes a single asymmetric tubule in non-dividing cells due to division of the kinetoplast portion

Kinetoplastid protozoa have a single mitochondrion that extends throughout the cell. The disk-shaped portion of the mitochondrion adjacent to the basal body of the flagellum contains the kinetoplast DNA nucleoid body which consists of thousands of catenated minicircles and a smaller number of catenated maxicircles. The maxicircles contain structural genes and cryptogenes, rRNA genes, and a few guide RNA genes The minicircles contain the majority of the guide RNA genes. The long slender non-dividing stationary phase Leishmania tarentolae cells in culture have an asymmetric mitochondrion that consists of a single tubule extending from one edge of the kinetoplast portion. This presents a problem for cell division, in that one daughter cell will receive significantly less mitochondrial membranes than the other cell. We show in this paper that the solution to this problem is that dividing cells, which are normally shorter and rounder than stationary phase cells, possess a symmetric circular mitochondrion that has mitochondrial tubules extending from both edges of the kinetoplast which are joined in the posterior region of the cell. This implies that growth of the mitochondrion occurs after cell division, either from elongation of the longitudinal tubule towards the anterior of the cell, or from elongation of the kinetoplast portion of the mitochondrion towards the posterior region and fusion of the tubules.

This work was performed by Larry Simpson and Frank Kretzer.

Lack of evidence for presence of 5' extentions on tRNAs imported into the mitochondrion of Leishmania

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in nuclear DNA and transported into the mitochondrion (Simpson et al., 1989, Nucl. Acids Res. 17: 5427-5445). It has been proposed by Hancock et al. (J. Biol. Chem., 1990, 265: 19208-19215) that tRNAs in these cells are imported into the mitochondrion as 5'-extended precursors which are processed by a mitochondrial RNase P-like activity. We have examined this hypothesis by cloning and sequencing primer extension products of mitochondrial tRNAs from L. tarentolae and T. brucei, and have found that these are derived from circularized mature tRNA molecules. We suggest that these molecules are produced by the endogenous RNA ligase activity either in vivo or during mitochondrial isolation. We did not obtain any evidence for the existence of high molecular weight precursors of mitochondrial tRNAs. This negative result is consistent with previous in vivo transfection studies with both L. tarentolae (Lima and Simpson, 1996, RNA 2: 429-440) and T. brucei (Hauser and Schneider, 1995, EMBO J. 14: 4212-4220), in which mitochondrial targeting of plasmid-expressed tRNAs was independent of the presence of 5'-flanking sequences. We conclude that the hypothesis for 5'-extended tRNA precursors in kinetoplastid mitochondrial importation remains to be verified.

This work was performed by Ruslan Aphasizhev.

Cloning and characterization of the Leishmania tarentolae adenine phosphoribosyltransferase

Adenine phosphoribosyltransferase (APRT) is an important enzyme involved in the recycling of purine nucleotides in all cells. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and utilize the salvage pathway for synthesis of ribonucleotides. The aprt gene was cloned from a Leishmania tarentolae genomic library and the sequence determined. The L. tarentolae aprt gene contains a 708 nucleotide open reading frame that encodes a 25 kDa protein. The predicted amino acid sequence has 85% identity to the APRT of L. donovani (Allen,T., Hwang, H., Wilson, K., Hanson, S., Jardim, A. and Ullman, B. (1995) Mol. Biochem. Parasitol. 74, 99-103). A recombinant protein was expressed in Escherichia coli, purified to homogeneity and found to retain enzymatic activity. The L. tarentolae APRT is active as a homodimer in solution. The availability of the APRT enzyme from another kinetoplastid protozoan and the possibility of expressing the recombinant protein in large quantities should provide the basis for a functional and structural analysis of this enzyme which has been suggested as a potential target for rational drug design.

This work was performed by Otavio Thiemann.

Purification and Characterization of MAR1: a Mitochondrial Associated Ribonuclease from Leishmania tarentolae

A relatively thermostable 22 kDa endoribonuclease (MAR1) was purified more than 10,000 fold from a mitochondrial extract of Leishmania tarentolae and the gene cloned. The purified nuclease has a Km of 100-145 ± 33 nM and a Vmax of 1.8-2.9 ± 2 nmoles/min, depending on the RNA substrate, and yields a 3' OH and a 5' phosphate. Cleavage was limited to several specific sites in the substrate RNAs tested, but cleavage of pre-edited RNAs was generally independent of the addition of cognate guide RNA. The MAR1 gene was expressed in Escherichia coli or in Leishmania tarentolae cells and the recombinant protein was affinity-purified. The cleavage specificity of the recombinant enzyme from Leishmania tarentolae was identical to that of the native enzyme. The single copy MAR1 gene maps to an 820 kb chromosome and contains an open reading frame of 579 nt. The 18 amino acid N-terminal sequence shows characteristics of an uncleaved mitochondrial targeting sequence. Database searching revealed two homologues of MAR1 corresponding to unidentified open reading frames in Caenorhabditis elegans (Z69637) and Archaeoglobus fulgidus (AE000943). The function of MAR1 in mitochondrial RNA metabolism in L. tarentolae remains to be determined. Click here and here to see the figures from this paper.

This work was performed by Juan Alfonzo and Otavio Thiemann.

Phylogenetic Affinities of Diplonema within the Euglenozoa as Inferred from the SSU rRNA Gene and Partial COI Protein Sequences

In order to shed light on the phylogenetic position of diplonemids within the phylum Euglenozoa, we have sequenced small subunit rRNA (SSU rRNA) genes from Diplonema (syn. Isonema) papillatum and Diplonema sp. We have also analyzed a partial sequence of the mitochondrial gene for cytochrome c oxidase subunit I from D. papillatum. With both markers, the maximum likelihood method favored a closer grouping of diplonemids with kinetoplastids, while the parsimony and distance suggested a closer relationship of diplonemids with euglenoids. In each case, the differences between the best tree and the alternative trees were small. The frequency of codon usage in the partial D. papillatum COI was different from both related groups; however, as is the case in kinetoplastids but not in Euglena, both the non-canonical UGA codon and the canonical UGG codon were used to encode tryptophan in Diplonema.

This research was performed by Dmitri Maslov and Shingi Yasuhira.

In vitro uridine insertion RNA editing mediated by cis-acting guide RN

Guide RNAs involved in mediating RNA editing in vivo are provided in trans except for the CO2 mRNA, which has the gRNA in cis at the 3' end of the RNA. We have found that a cognate gRNA provided in cis at the 3' end of a pre-edited NADH dehydrogenase 7 (ND7) mRNA substrate can direct U insertions at editing site 1 when incubated with a mitochondrial lysate from Leishmania tarentolae. The efficiency of gRNA-dependent U insertion mediated by a cis-acting is greater on a molar basis than that for a trans-acting gRNA, as expected for a unimolecular gRNA:mRNA interaction. Blocking the 3' end of a cis-acting gRNA lacking a 3' oligo[U] tail has no effect on gRNA-dependent U insertions, nor does providing the gRNA in cis upstream of the mRNA, confirming the previous observation that the terminal 2'- and 3'-hydroxyls of the gRNA are not involved in U insertion activity. These results also establish that the oligo[U] tail is not required for U insertion in vitro. Increasing the extent of base pairing between the 3' end of the gRNA and the 5' end of the mRNA significantly increases in vitro gRNA-dependent U insertion at site 1, presumably by maintaining the mRNA 5' cleavage fragment within the editing complex. We speculate that, in vivo, protein:RNA and/or protein:protein interactions may be responsible for maintaining the mRNA 5' cleavage fragment in close proximity to the mRNA 3' cleavage fragment, and that such interactions may be rate limiting in vitro.

This work was done by Steve Kapuschoc.

The Mitochondrial RNA ligase from Leishmania tarentolae can join RNA molecules bridged by a complementary RNA

A biochemical characterization was performed with a partially purified RNA ligase from isolated mitochondria of Leishmania tarentolae. This ligase has a Km of 25 ± 0.75 nM and a Vmax of 1.0 x10-4 ± 2.4 x 10-4 nmoles/min when ligating a nicked double stranded RNA substrate. Ligation was negatively affected by a gap between the donor and acceptor nucleotides. The catalytic efficiency of the circularization of a single stranded substrate was five-fold less than that of the ligation of a nicked substrate. These properties of the mitochondrial RNA ligase are consistent with an expected in vivo role in the process of uridine insertion/deletion RNA editing, in which the mRNA cleavage fragments are bridged by a cognate guide RNA.

Click here to see the figures from this paper.

This work was done by Valerie Blanc, Juan D. Alfonzo, and Ruslan Aphasizhev.

T7 RNA polymerase-driven transcription in mitochondria of Leishmania tarentolae and Trypanosoma brucei.

The study of RNA editing and other molecular processes in the trypanosome mitochondrion would benefit greatly from the ability to insert and express exogenous DNA in the organelle. However, even with a method to introduce DNA, the current lack of knowledge about mitochondrial transcription would hinder efforts to obtain expression. To circumvent this problem, we have transfected Leishmania tarentolae promastigotes and Trypanosoma brucei procyclic cells with bacteriophage T7 RNA polymerase targeted to the mitochondrion. Mitochondria isolated from the transfectants contained active T7 RNA polymerase, as shown by a comigration in density gradients of mitochondrial marker enzymes and T7 polymerase activity. A DNA cassette under T7 control was introduced into isolated mitochondria from the transfectants by electroporation and the DNA was shown to be transcribed. This system should allow the transcription of foreign genes of choice within the mitochondrial matrix either in a transient assay using electroporation of DNA into isolated mitochondria, or in a stable assay using cells transfected with DNA by the biolistic gun method.

Click here to see the figures from this work.

This work was done by Antonio M. Estévez, Otavio H. Thiemann and Juan D. Alfonzo.

C to U editing of Anticodon of Imported Mitochondrial tRNATrp allows Decoding of UGA Stop Codon in Leishmania tarentolae

All mitochondrial tRNAs in kinetoplastid protists are encoded in the nucleus and imported into the organelle. The tRNATrp(CCA) can decode the standard UGG tryptophan codon but cannot decode the mitochondrial UGA tryptophan codon. We show that the mitochondrial tRNATrp undergoes a specific C to U nucleotide modification in the first position of the anticodon which allows decoding of mitochondrial UGA codons as tryptophan. Functional evidence for the absence of a UGA suppressor tRNA in the cytosol, using a reporter gene, was also obtained, which is consistent with a mitochondrial localization of this editing event. Leishmania cells have dealt with the problem of a lack of expression within the organelle of this non-universal tRNA by compartmentalizing an editing activity which modifies the anticodon of the imported tRNA.

This work was performed by Juan D. Alfonzo, Valerie Blanc, Antonio M. Estévez and Mary Anne T. Rubio.

Evolution of RNA Editing in Trypanosome Mitochondria

Two different RNA editing systems have been described in the kinetoplast-mitochondrion of trypanosomatid protists. The first involves the precise insertion and deletion of U residues mostly within the coding regions of maxicircle-encoded mRNAs to produce open reading frames. This editing is mediated by short overlapping complementary guide RNAs (gRNAs) encoded in both the maxicircle and the minicircle molecules, and involves a series of enzymatic cleavage-ligation steps. The second editing system is a C34 to U34 modification in the anticodon of the imported tRNATrp, thereby permitting the decoding of the UGA stop codon as tryptophan. U-insertion editing probably originated in an ancestor of the kinetoplastid lineage and has evolved in some cases by the replacement of the original pan-edited cryptogene with a partially edited cDNA. The driving force for this retroposition was postulated to be the stochastic loss of entire minicircle sequence classes and their encoded gRNAs upon segregation of the single kinetoplast DNA network into daughter cells at cell division. A large plasticity in frequencies of minicircle sequence classes has been observed during cell culture in the laboratory. Computer simulations provide theoretical evidence for this plasticity if a random distribution and segregation model of minicircles is assumed. The specific C to U tRNA editing probably evolved after the loss of the original endogenous tRNA genes from the mitochondrial genome and the development of a mechanism for importation of nuclear-encoded tRNAs into the organelle. The relationship of the two editing systems is discussed.

This work was performed by Otavio Thiemann, Nickloas Savill, Juan Alfonzo, and Dmitri Maslov. It was presented at the Colloquium on Variation and Evolution in Plants and Microorganisms, Jan. 27-29, 2000, Beckman Center of the National Academies of Sciences and Engineering, Irvine, CA.

Selective Importation of RNA into Isolated Mitochondria from Leishmania tarentolae

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro transcribed tRNAs, tRNAIle(UAU) and tRNAGln(CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNAAsp was protected from nuclease digestion and was also cleaved by an intra-mitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than approximately 17 nt, and that smaller RNAs are apparently non-specifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondria-localized tRNAIle as compared to the level of importation of the mainly cytosol-localized tRNAGln. Furthermore, exchanging the D-arm between the tRNAIle and the tRNAGln resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.

This work was performed by Mary Anne T. Rubio, Xuan Liu, Harumi Yuzawa and Juan D. Alfonzo.

End processing precedes mitochondrial importation and editing of tRNAs in Leishmania tarentolae

All mitochondrial tRNAs in Leishmania tarentolae are encoded in the nuclear genome and imported into the mitochondrion from the cytosol. One imported tRNA (tRNATrp) is edited by a C to U modification at the first position of the anticodon. In order to determine the in vivo substrates for mitochondrial tRNA importation as well as tRNA editing, we examined the subcellular localization and extent of 5'- and 3'-end maturation of tRNATrp(CCA), tRNAIle(UAU), tRNAGln(CUG), tRNALys(UUU), and tRNAVal(CAC). Nuclear, cytosolic and mitochondrial fractions were obtained with little cross contamination, as determined by northern analysis of specific marker RNAs. The tRNAGln was mainly cytosolic in localization, the tRNAIle and tRNALys were mainly mitochondrial, and the tRNATrp and tRNAVal were shared between the two compartments. 5'- and 3'-extended precursors of all five tRNAs were present only in the nuclear fraction, suggesting that the mature tRNAs represent the in vivo substrates for importation into the mitochondrion. Consistent with this model, T7-transcribed mature tRNAIle undergoes importation in vitro into isolated mitochondria more efficiently than the 5'-extended precursor tRNAIle. The 5'-extended precursor tRNATrp was found to be unedited, which is consistent with a mitochondrial localization of this editing reaction. T7-transcribed unedited tRNATrp was imported in vitro more efficiently than edited tRNATrp, suggesting the presence of importation determinants in the anticodon.

This research was performed by Steve Kapushoc, Juan Alfonzo and Mary Anne Rubio.

Uridine insertion/deletion RNA editing in L. tarentolae mitochondria shows cell cycle dependence

L. tarentolae cells were synchronized using hydroxyurea and the relative abundance of edited and pre-edited transcripts for 4 maxicircle genes was analyzed by primer extension. The primers in each case hybridized to unedited sequence downstream of the editing domain, so that both unedited and edited extension products can be observed on the same gel. The FE/UE ratio was found to vary from 1.3 to 2.0 fold for all four genes during the cell cycle. The ratio peaked in S+G1 and then again in the same phase of the second synchronized cycle. This variation is most likely due to variation in the extent of editing. The level of this regulation is not known. This phenomenon may have some relationship to the synchronicity of nuclear and mitochondrial replication phases, which appears to involve a differential expression of nuclear-encoded replication proteins due to differential turnover of mRNAs (see papers from Dan Ray lab).

This work was performed by R. Carrillo, O. Thiemann and J. Alfonzo.

Isolation and Characterization of a U-specific 3'-5' Exonuclease from Mitochondria of Leishmania tarentolae

We have purified a 3'-5' exoribonuclease from mitochondrial extract of Leishmania tarentolae over 4000-fold through six column fractionations. This enzyme digested RNA in a distributive manner, showed a high level of specificity for 3' terminal U's and was blocked by a terminal dU or pCp; there was a slight exonucleolytic activity on a 3' terminal A or C, but no activity on a 3' terminal G residue. The enzyme preferred single-stranded oligo[U] 3' and did not digest duplex RNA. Two other 3'-5' exoribonuclease activities were also detected in the mitochondrial extract, one of which was stimulated by a 3'-phosphate and the other of which degraded RNAs with a 3'OH to mononucleotides in a processive manner. The properties of the distributive U-specific 3'-5' exoribonuclease suggest an involvement in the U-deletion RNA editing reaction that occurs in the mitochondrion of these cells.

This work was performed by Ruslan Aphasizhev.

Guide RNAs of the recently isolated LEM 125 strain of Leishmania tarentolae: an unexpected complexity

Guide RNAs (gRNAs) are encoded both in the maxicircle and minicircle components of the mitochondrial DNA of trypanosomatid protozoa. These RNAs mediate the precise insertion and deletion of U residues in transcripts of the maxicircle DNA. We showed previously that the old UC laboratory strain of Leishmania tarentolae apparently lost more than 40 minicircle-encoded gRNAs which are present in the recently isolated LEM125 strain (Thiemann et al., 1994). We have further analyzed the population of minicircle-encoded gRNAs in the LEM125 strain. Sau3AI and MspI minicircle libraries were constructed and screened for novel gRNAs by negative colony hybridization. This search yielded 20 minicircles encoding new gRNAs that covered most of the remaining gaps in the editing cascades of the ND8, ND9, G3 and G4 genes, and in addition, more than 30 minicircles containing either unassigned or undetectable gRNA genes. We also completely sequenced 34 of the 45 minicircle sequence classes encoding previously identified gRNAs. A total of 19 pairs of redundant gRNAs, which are gRNAs of different sequences covering the same editing blocks, were identified. The redundant gRNA pairs showed a differential steady-state abundance and mismatches may represent one of the factors determining this abundance differences. Alignments of the minicircles encoding redundant gRNAs yielded 59 to 93% matching nucleotides, suggesting an origin from duplication of ancestral minicircles and subsequent genetic drift. We propose a functional explanation for the existence of redundant gRNAs in this strain.

Differential localization of nuclear-encoded tRNAs between the cytosol and mitochondrion in Leishmania tarentolae

All mitochondrial tRNAs of the kinetoplastid protozoan Leishmania tarentolae are encoded in the nucleus and are imported from the cytosol into the mitochondrion. We previously reported the partitioning of five tRNAs and found that all were shared between the two compartments to different extents. In order to increase our knowledge of the tRNAs of this organism, and to attempt to understand the signals involved in their subcellular localization, a method to RT-PCR amplify new tRNAs was developed. Various tRNAs were 3' polyadenylated and reverse transcribed with a sequence tagged primer. The cDNA was tagged by ligation to an anchor oligonucleotide, and the resulting double-tagged cDNA was amplified by PCR. Four new tRNAs were obtained, bringing to 20 the total number of L. tarentolae tRNAs identified to date. The subcellular localization of 17 tRNAs was quantitatively analyzed by two-dimensional gel electrophoresis and Northern hybridization. In general, the previously suggested operational classification of tRNAs into three groups (mainly cytosolic, mainly mitochondrial, and shared between the two compartments) is still valid, but the relative abundance of each tRNA in the cytosol or mitochondrion varies greatly as does the level of expression.

This work was performed by Steve Kapushoc and Juan Alfonzo.

Trypanosome Mitochondrial 3' Terminal Uridylyl Transferase (TUTase): A Key Enzyme in U-insertion/deletion RNA Editing

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++. TUTase is present in vivo in at least two stable configurations: One contains a ~500 kDa TUTase oligomer and the other a ~700 kDa TUTase complex. Anti-TUTase antiserum specifically co-precipitates 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 down-regulates RNA editing and appears to affect parasite viability.

This work was performed by Ruslan Aphasizhev et al.

Modification of the universally unmodified uridine-33 in a mitochondria-imported edited tRNA and the role of the anticodon arm structure on editing efficiency

Editing of tRNA has a wide phylogenetic distribution among eukaryotes and in some cases serves to expand the decoding capacity of the target tRNA. We previously described C to U editing of the wobble position of the imported tRNATrp in Leishmania mitochondria which is essential for decoding UGA codons as tryptophan. Here we show the complete set of nucleotide modifications in the anticodon arm of the mitochondrial and cytosolic tRNATrp as determined by electrospray ionization mass spectrometry. This analysis revealed extensive mitochondria-specific post-transcriptional modifications, including the first example of thiolation of U33, the "universally unmodified" uridine. In light of the known rigidity imparted on sugar conformation by thiolation, our discovery of a thiolated U33 suggests that conformational flexibility is not a universal feature of the anticodon structural signature. In addition, the in vivo analysis of tRNATrp variants presented shows a single base pair reversal in the anticodon stem of tRNATrp is sufficient to abrogate editing in vivo, indicating that subtle changes in anticodon structure can have drastic effects on editing efficiency.

This work was performed by Crain, P.F., Alfonzo, J.D., Rozenski, J., Kapushoc, S.T., McCloskey, J.A. and Simpson, L.

Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism

In Leishmania tarentolae, all the tRNAs required for mitochondrial translation are encoded in the nuclear genome and imported from the cytosol. It is known that tRNAGlu(UUC) and tRNAGln(UUG) are localized in both cytosol and mitochondria. We investigated structural differences between affinity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for Glu and Gln by direct enzymatic sequencing and mass spectrometric analysis. A unique modification difference in both tRNAs was identified at the anticodon wobble position: cy tRNAs have 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), whereas mt tRNAs have 5- methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm5U) at its wobble position, which could represent a common modification intermediate for both modified uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-specific tRNALys(UUU) from the cytosol and found mcm5U at its wobble position, while its mitochondrial counterpart has mcm5Um. We found that mt tRNALys and in vitro-transcribed tRNAGlu were imported much more efficiently into isolated mitochondria than the native cy tRNAGlu in an in vitro importation assay, indicating that cytosol specific 2-thiolation could play an inhibitory role in tRNA import into mitochondria.

This work is the result of a collaboration and was performed by Tomonori Kaneko, Takeo Suzuki, Stephan T. Kapushoc, Mary Anne Rubio, Jafar Ghazvini, Kimitsuna Watanabe and Tsutomu Suzuki

A 100 kDa complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components

A stable 100 kD complex from mitochondria of Leishmania tarentolae containing two RNA-binding proteins, Ltp26 and Ltp28, was identified by cross-linking to unpaired 4-thiouridine nucleotides in a partially duplex RNA substrate. The genes were cloned and expressed and the complex was reconstituted from recombinant proteins in the absence of RNA or additional factors. The Ltp26 and Ltp28 proteins are homologues of gBP27 and gBP29 from Crithidia fasciculata, and gBP25 and gBP21 from Trypanosoma brucei, respectively. The purified Ltp26/Ltp28 complex, the individual recombinant proteins and the reconstituted complex are each capable of catalyzing the annealing of complementary RNAs, as was previously shown for gBP21 from T. brucei. A high molecular weight RNP complex consisting of the Ltp26/Ltp28 complex and several 55-60 kDa proteins together with guide RNA could be purified from mitochondrial extract of L. tarentolae transfected with Ltp28-TAP. This complex also interacted in a less stable manner with the RNA ligase-containing L- complex and with the 3' TUTase. The Ltp26/Ltp28 RNP complex is a candidate for catalyzing the annealing of guide RNA and pre-edited mRNA in the initial step of RNA editing.

Update 6-16-2003: These proteins are now labeled MRP1 and MRP2 and the complex is labeled MRP1/2.

This work was performed by R. Aphasizhev, I. Aphasizhev and R. Nelson.

Is the Trypanosoma brucei REL1 RNA ligase specific for U-deletion RNA editing and the REL2 RNA ligase specific for U-insertion editing?

It was shown previously that the REL1 mitochondrial RNA ligase in Trypanosoma brucei was a vital gene and disruption affected RNA editing in vivo whereas the REL2 RNA ligase gene could be down regulated with no effect on cell growth or on RNA editing. We performed down regulation of REL1 in procyclic T. brucei (midgut insect forms) by RNAi and found a 40-50% inhibition of Cyb editing, which has only U-insertions, as well as a similar inhibition of ND7 editing, which has both U-insertions and U-deletions. In addition, both U-insertion and U-deletion in vitro pre-cleaved editing were inhibited to similar extents. We also found little if any effect of REL1 down regulation on the sedimentation coefficient or abundance of the RNA ligase-containing L-complex (Aphasizhev et al. (2003) EMBO J. 22:913-924), suggesting that the inhibition of both insertion and deletion editing was not due to a disruption of the L-complex. Together with the evidence that down regulation of REL2 has no effect on cell growth or on RNA editing in vivo or in vitro, these data suggest that the REL1 RNA ligase may be active in vivo in both U-insertion and U-deletion editing. The in vivo biological role of REL2 remains obscure.

This work was performed by G. Gao.

Isolation of an RNA Editing Complex Active in Both U-insertion and U-deletion in vitro Editing

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 affnity purification (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 identified 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.

This figure shows the isolated L-complex sediments as a single band in a glycerol gradient and contains around 15 proteins.

This work was performed by Ruslan Aphasizhev, Inna Aphasizheva, Robert E.Nelson, Guanghan Gao, Agda M.Simpson, Xuedong Kang, Arnold M.Falick, and Sandro Sbicego

RBP38, an RNA-binding protein from trypanosomatid mitochondria, modulates RNA stability

A novel RNA-binding protein, RBP38, was isolated 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 RNA substrates with dissociation constants in the 100 nanomolar range, as determined by fluorescence polarization analysis. Down regulation of expression of the homologous gene, TbRBP38, in procyclic T. brucei using conditional RNAi 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 ~160 min to ~60 min after depletion. In contrast, there was no change in transcriptional activity. These observations suggest a role of RBP38 in stabilizing mitochondrial RNA.

This work was performed by Sandro Sbicego, Juan D. Alfonzo, Antonio M. Estévez, Mary Anne T. Rubio, Xuedong Kang, Christoph W. Turck and Marian Peris

Genomic Organization of Trypanosoma brucei Kinetoplast DNA Minicircles

The sequences of seven new Trypanosoma brucei kinetoplast DNA minicircles were obtained. A detailed comparative analysis of these sequences and those of the 18 complete kDNA minicircle sequences from T. brucei available in the database was performed. These 25 different minicircles contain 86 putative gRNA genes. The number of gRNA genes per minicircle varies from 2 to 5. In most cases, the genes are located between short imperfect inverted repeats, but in several minicircles there are inverted repeat cassettes that did not contain identifiable gRNA genes. Five minicircles contain single gRNA genes not surrounded by identifiable repeats. Two pairs of closely related minicircles may have recently evolved from common ancestors: KTMH1 and KTMH3 contained the same gRNA genes in the same order, whereas KTCSGRA and KTCSGRB contained two gRNA genes in the same order and one gRNA gene specific to each. All minicircles could be classified into two classes on the basis of a short substitution within the highly conserved region, but the minicircles in these two classes did not appear to differ in terms of gRNA content or gene organization. A number of redundant gRNAs containing identical editing information but different sequences were present. The alignments of the predicted gRNAs with the edited mRNA sequences varied from a perfect alignment without gaps to alignments with multiple mismatches. Multiple gRNAs overlapped with upstream gRNAs, but in no case was a complete set of overlapping gRNAs covering an entire editing domain obtained. We estimate that a minimum set of approximately 65 additional gRNAs would be required for complete overlapping sets. This analysis should provide a basis for detailed studies of the evolution and role in RNA editing of kDNA minicircles in this species.

This work was done by Min Hong.

The RET1 TUTase adds U's to the gRNA 3' end and the RET2 TUTase adds U's at editing sites of mRNAs

We have described two TUTases, RET1 (RNA Editing TUTase 1) and RET2 (RNA Editing TUTase 2) as components of different editing complexes. TAP-tagged Trypanosoma brucei RET2 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 uridine 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 U-insertion 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 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 relabelled the previously described TUT-II complex containing RET1 as the GP-complex (Guide RNA Processing).

This work was done by Ruslan Aphasizhev and Inna Aphasizheva.

Loss of editing due to extended culture of cells. Larry Simpson, Stephen M. Douglass, James A. Lake,Matteo Pellegrini and Feng Li We analyzed by NGS technology the mitochondrial genomes and transcriptomes of two strains, the old lab UC strain and the recently isolated LEM125 strain. PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions. Minicircles were identified by a characteristic size, the presence of three short conserved sequences, a region of inherently bent DNA and the presence of single gRNA genes at a fairly defined location. The LEM125 strain contained over 114 minicircles encoding different gRNAs and the UC strain only ~24 minicircles. Some LEM125 minicircles contained no identifiable gRNAs. Approximate copy numbers of the different minicircle classes in the network were determined by the number of PacBio CCS reads that assembled to each class. Mitochondrial RNA libraries from both strains were mapped against the minicircle and maxicircle sequences. Small RNA reads mapped to the putative gRNA genes but also to multiple regions outside the genes on both strands and large RNA reads mapped in many cases over almost the entire minicircle on both strands. These data suggest that minicircle transcription is complete and bidirectional, with 3’ processing yielding the mature gRNAs. Steady state RNAs in varying abundances are derived from all maxicircle genes, including portions of the repetitive divergent region. The relative extents of editing in both strains correlated with the presence of a cascade of cognate gRNAs. U-insertion/deletion RNA editing multiprotein complexes and mitochondrial ribosomes in Leishmania tarentolae are located in antipodal nodes adjacent to the kinetoplast DNA Richard G. Wong, Katelynn Kazane, Dmitri A. Maslov, Kestrel Rogers, Ruslan Aphasizhev and Larry Simpson We studied the intramitochondrial localization of several multiprotein complexes involved in U-insertion/deletion RNA editing in trypanosome mitochondria. The editing complexes are located in one or two antipodal nodes adjacent to the kinetoplast DNA (kDNA) disk, which are distinct from but associated with the minicircle catenation nodes. In some cases the proteins are in a bilateral sheet configuration. We also found that mitoribosomes have a nodal configuration. This type of organization is consistent with evidence for protein and RNA interactions of multiple editing complexes to form an ~40S editosome and also an interaction of editosomes with mitochondrial ribosomes