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PubMed2005

Unique mitochondrial genome structure in diplonemids, the sister group of kinetoplastids.

Kinetoplastid flagellates are characterized by uniquely massed mitochondrial DNAs (mtDNAs), the kinetoplasts. Kinetoplastids of the trypanosomatid group possess two types of mtDNA molecules: maxicircles bearing protein and mitoribosomal genes and minicircles specifying guide RNAs, which mediate uridine insertion/deletion RNA editing. These circles are interlocked with one another to form dense networks. Whether these peculiar mtDNA features are restricted to kinetoplastids or prevail throughout Euglenozoa (euglenids, diplonemids, and kinetoplastids) is unknown. Here, we describe the mitochondrial genome and the mitochondrial ultrastructure of Diplonema papillatum, a member of the diplonemid flagellates, the sister group of kinetoplastids. Fluorescence and electron microscopy show a single mitochondrion per cell with an ultrastructure atypical for Euglenozoa. In addition, DNA is evenly distributed throughout the organelle rather than compacted. Molecular and electron microscopy studies distinguish numerous 6- and 7-kbp-sized mitochondrial chromosomes of monomeric circular topology and relaxed conformation in vivo. Remarkably, the cox1 gene (and probably other mitochondrial genes) is fragmented, with separate gene pieces encoded on different chromosomes. Generation of the contiguous cox1 mRNA requires trans-splicing, the precise mechanism of which remains to be determined. Taken together, the mitochondrial gene/genome structure of Diplonema is not only different from that of kinetoplastids but unique among eukaryotes as a whole.

Animals↗
PubMed1990

A model for RNA editing in kinetoplastid mitochondria: "guide" RNA molecules transcribed from maxicircle DNA provide the edited information.

A class of small RNA molecules possibly involved in RNA editing is present in the mitochondrion of Leishmania tarentolae. These "guide" RNA (gRNA) molecules are encoded in intergenic regions of the mitochondrial maxicircle DNA and contain sequences that represent precise complementary versions of the mature mRNAs within the edited regions. In addition, the 5' portions of several gRNAs can form hybrids with mRNAs just 3' of the preedited region. A model is presented in which a partial hybrid formed between the gRNA and preedited mRNA is substrate for multiple cycles of cleavage, addition or deletion of uridylates, and religation, eventually resulting in a complete hybrid between the gRNA and the mature edited mRNA.

Animals↗
PubMed2005

Biphasic decay of guide RNAs in Trypanosoma brucei.

Guide RNAs (gRNAs) are short mitochondrially encoded RNAs that contain the information for editing of messenger RNAs in Trypanosoma brucei. Although a great deal of work has focused on the utilization of gRNAs in editing, little is known about the turnover of gRNAs. In this report, we utilized in organello pulse chase and in vitro RNA decay experiments to directly examine gRNA turnover. We found that gRNAs are degraded by a biphasic mechanism. In the first step of decay, 3' gRNA sequences encompassing primarily the post-transcriptionally added oligo(U) tail are rapidly removed. This is followed by a second step, which entails a comparatively slower degradation of the encoded gRNA body. Decay of the 3' end of the gRNA is sequence specific, as it does not occur on oligoadenylated gRNAs. In contrast, the nucleotide composition of the 3' extension does not affect the rate of degradation during the second, slower, decay step. Finally, competition assays suggest that complete gRNA decay is mediated by two distinct enzymes, one of which simultaneously recognizes elements of the oligo(U) tail and the encoded portion of the gRNA. Overall, these results provide the first evidence for a gRNA-specific decay pathway.

Animals↗
PubMed1998

The mitochondrial ND8 gene from Crithidia oncopelti is not pan-edited.

RNA editing in trypanosomatid mitochondria is a process involving the insertion and deletion of uridine residues within the coding region of maxicircle messenger RNA transcripts. Twelve of the 17 known genes need editing to produce functional molecules. We have analyzed the predicted editing sites for the Crithidia oncopelti mitochondrial NADH-ubiquinone oxidoreductase subunit 8 (ND8) gene based on known mRNAs from other trypanosomatid species. All studied ND8 mRNAs undergo editing throughout the coding (and 3' noncoding) sequences (pan-editing). The 5' part of the C. oncopelti ND8 gene undergoes editing (like in Leishmania tarentolae and Trypanosoma brucei) while the 3' part of the pre-edited gene corresponds to the 3' part of edited ND8 mRNAs from other organisms. The organization of the ND8 gene in C. oncopelti mitochondrial DNA differs from all organisms investigated so far -- this gene is not pan-edited. We have also localized the guide RNA for cytochrome b between 9S rRNA and the ND8 gene. This RNA shows high homology to the gCYb-II gene of L. tarentolae and the gCyb gene of Crithidia fasciculata. A hypothetical editing pattern for the cytochrome b gene in C. oncopelti maxicircles is proposed.

Amino Acid Sequence↗
PubMed2000

Redundant and non-functional guide RNA genes in Trypanosoma brucei are a consequence of multiple genes per minicircle.

The mitochondrial mRNA of the parasitic protozoa Trypanosoma brucei is extensively edited by the insertion, and occasional deletion, of uridine residues. The editing is mediated by over 200 guide RNAs (gRNAs) that are encoded in circular DNA molecules called minicircles. There are some 250 different types of minicircle, called classes, with each encoding several gRNAs. Sequencing of gRNAs and minicircles has revealed a surprising amount of both redundancy, where gRNAs from different minicircle classes edit exactly the same part of an mRNA, and non-functionality, where partial or no complementarity is found between gRNA and mRNA. How does this redundancy and non-functionality arise and persist? We propose the following. Minicircle classes that contain several functional gRNA genes can be lost from the population via drift and replaced by more minicircle classes that contain fewer functional gRNA genes, on the condition that the cells keep a full complement of functional gRNAs. We demonstrate this hypothesis in a computer simulation of a model of minicircle evolution. We show that this process leads to an increasing number of minicircle classes and inevitably to only one functional gRNA per minicircle. Moreover, we show that the genome contains more minicircle classes than is actually necessary for cell survival. We also analyse the available minicircle sequence data and conclude that T. brucei is at a transient stage in this process. In addition, ten new putative gRNAs have been discovered.

Animals↗
PubMed2000

Mitochondrial minicircles in the free-living bodonid Bodo saltans contain two gRNA gene cassettes and are not found in large networks.

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T. borreli. We identified 1.4-kb circular DNAs as the B. saltans equivalent of minicircles via sequence analysis of 4 complete minicircles, 14 minicircle fragments, and 14 gRNAs. We show that each minicircle harbors two gRNA gene cassettes of opposite polarity residing in variable regions of about 200 nt in otherwise highly conserved molecules. In the conserved region, B. saltans minicircles contain a putative bent helix sequence and a degenerate dodecamer motif (CSB-3). Electron microscopy, sedimentation, and gel electrophoresis analyses showed no evidence for the existence of large minicircle networks in B. saltans, the large majority of the minicircles being present as circular and linear monomers (85-90%) with small amounts of catenated dimers and trimers. Our results provide the first example of a kinetoplastid species with noncatenated, gRNA gene-containing minicircles, which implies that the creation of minicircles and minicircle networks are separate evolutionary events.

Amino Acid Sequence↗
PubMed2001

Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei.

Most mitochondrial genes of Trypanosoma brucei do not contain the necessary information to make translatable mRNAs. These transcripts must undergo RNA editing, a posttranscriptional process by which uridine residues are added and deleted from mitochondrial mRNAs. RNA editing is believed to be catalyzed by a ribonucleoprotein complex containing endonucleolytic, terminal uridylyl transferase (TUTase), 3' uridine-specific exonucleolytic (U-exo), and ligase activities. None of the catalytic enzymes for RNA editing have been identified. Here we describe the identification of two candidate RNA ligases (48 and 52 kDa) that are core catalytic components of the T. brucei ribonucleoprotein editing complex. Both enzymes share homology to the covalent nucleotidyl transferase superfamily and contain five key signature motifs, including the active site KXXG. In this report, we present data on the proposed 48 kDa RNA editing ligase. We have prepared polyclonal antibodies against recombinant 48 kDa ligase that specifically recognize the trypanosome enzyme. When expressed in trypanosomes as an epitope-tagged fusion protein, the recombinant ligase localizes to the mitochondrion, associates with RNA editing complexes, and adenylates with ATP. These findings provide strong support for the enzymatic cascade model for kinetoplastid RNA editing.

Adenosine Triphosphate↗
PubMed1993

The boundaries of partially edited transcripts are not conserved in kinetoplastids: implications for the guide RNA model of editing.

We have studied partially edited molecules for the cytochrome-c oxidase subunit III (COIII) transcript from two species of the insect trypanosome Herpetomonas. We found unexpected patterns of editing, in which editing does not proceed strictly 3' to 5', in 24 of 61 partially edited clones. A comparison of the partially edited molecules between the two kinetoplastid species revealed an 8- to 10-nt shift in precisely defined editing boundaries, sites at which editing pauses before binding of the next guide RNA after formation of a stable duplex between a guide RNA and mRNA. This suggests that the region of base pairing between individual guide RNAs and the COIII transcript is not strictly conserved in kinetoplastids, implying gradual evolution of the editing process.

Animals↗
PubMed2003

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.

Animals↗
PubMed1998

The guide RNA database.

Guide RNAs (gRNAs) are small, metabolically stable RNA molecules which perform a pivotal, template-like function during the RNA editing process in kinetoplastid protozoa. The gRNA database currently contains 250 guide RNA sequences as well as secondary and tertiary structure models and other relevant information. The database is made available as a hypertext document accessible via the World Wide Web (WWW) at the URL: http://www.biochem.mpg.de/ goeringe/

Base Sequence↗
PubMed1993

Group II intron RNA catalysis of progressive nucleotide insertion: a model for RNA editing.

The self-splicing bl1 intron lariat from mitochondria of Saccharomyces cerevisiae catalyzed the insertion of nucleotidyl monomers derived from the 3' end of a donor RNA into an acceptor RNA in a 3' to 5' direction in vitro. In this catalyzed reaction, the site specificity provided by intermolecular base pair interactions, the formation of chimeric intermediates, the polarity of the nucleotidyl insertion, and its reversibility all resemble such properties in previously proposed models of RNA editing in kinetoplastid mitochondria. These results suggest that RNA editing occurs by way of a concerted, two-step transesterification mechanism and that RNA splicing and RNA editing might be prebiotically related mechanisms; possibly, both evolved from a primordial demand for self-replication.

Adenosine↗
PubMed2004

An intragenic guide RNA location suggests a complex mechanism for mitochondrial gene expression in Trypanosoma brucei.

In Trypanosoma brucei, two classes of transcripts are produced from two distinct mitochondrial genome components. Guide RNAs (gRNAs) are usually minicircle encoded and exist as primary transcripts, while the maxicircle-encoded rRNAs and mRNAs are processed from a polycistronic precursor. The genes for the gRNAs gMURF2-II and gCYb(560) each have uncommon kinetoplast DNA (kDNA) locations that are not typically associated with transcription initiation events. We demonstrate that the conserved maxicircle gRNA gMURF2-II has an unusual location within the ND4 gene. This is the first report of a completely intragenic gene in kDNA. In addition, the gMURF2-II and ND4 transcripts are generated by distinctly different events; the ND4 mRNA is processed from a polycistronic precursor, while transcription of the gRNA initiates downstream of the 5' end of the ND4 gene. The gCYb(560) gene has an atypical minicircle location in that it is not flanked by the inverted repeat sequences that surround the majority of minicircle gRNA genes. Our data indicate that the mature gCYb(560) gRNA is also a primary transcript and that the 5'-end heterogeneity previously observed for this gRNA is a result of multiple transcription initiation sites and not of imprecise 5'-end processing. Together, these data indicate that gRNA genes represent individual transcription units, regardless of their genomic context, and suggest a complex mechanism for mitochondrial gene expression in T. brucei.

Animals↗
PubMed1994

Ancient origin of RNA editing in kinetoplastid protozoa.

A rooted phylogenetic tree of the kinetoplastid protozoa has been constructed that, together with a comparative analysis of editing of several genes, leads to the surprising conclusion that extensive or pan-editing with multiple overlapping guide RNAs is more ancient than 5'-editing. The mechanism of editing is still uncertain, but multiple ribonucleoprotein complexes have been identified that contain components of the enzymatic machinery.

Animals↗
PubMed1997

RNA editing in kinetoplastid protozoa.

Mitochondrial transcripts in kinetoplastids undergo remarkable posttranscriptional editing by uridylate insertion and deletion. The often dramatic remodeling of pre-mRNA sequences is directed by small guide RNAs (gRNAs) to produce mature mRNAs. In vitro analyses of editing have been used to determine the mechanism of editing and show that editing occurs by a series of enzyme-catalyzed steps. They also show that chimeric gRNA/mRNA molecules are not editing intermediates as proposed but are aberrant end products of editing. The complexes and molecules that catalyze editing are now being identified and characterized. The origin of editing, its developmental regulation which helps control the switching between terminal respiratory systems during the life cycle of trypanosomes, and other areas for future study are discussed.

Animals↗
PubMed2004

The structure and replication of kinetoplast DNA.

Kinetoplast DNA (kDNA), the mitochondrial DNA of flagellated protozoa of the order Kinetoplastida, is unique in its structure, function and mode of replication. It consists of few dozen maxicircles, encoding typical mitochondrial proteins and ribosomal RNA, and several thousands minicircles, encoding guide RNA molecules that function in the editing of maxicircles mRNA transcripts. kDNA minicircles and maxicircles in the parasitic species of the family Trypanosomatidae are topologically linked, forming a two dimensional fishnet-type DNA catenane. Studies of early branching free-living and parasitic species of the Bodonidae family revealed various other forms of this remarkable DNA structure and suggested the evolution of kDNA from unlinked DNA circles and covalently-linked concatamers into a giant topological catenane. The replication of kDNA occurs during nuclear S phase and includes the duplication of free detached minicircles and catenated maxicircle and the generation of two progeny kDNA networks that segregate upon cell division. Recent reports of sequence elements and specific proteins that regulate the periodic expression of replication proteins advanced our understanding of the mechanisms that regulate the temporal link between mitochondrial and nuclear DNA synthesis in trypanosomatids. Studies on kDNA replication enzymes and binding proteins revealed their remarkable organization in clusters at defined sites flanking the kDNA disk, in correlation with the progress in the cell cycle and the process of kDNA replication. In this review I describe the recent advances in the study of kDNA and discuss some of the major challenges in deciphering the structure, replication and segregation of this remarkable DNA structure.

Animals↗
PubMed2003

Cis-acting elements stimulating kinetoplastid guide RNA-directed editing.

The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created through the insertion and deletion of specific uridylates. The editing reactions are required to be highly specific in order to ensure that functional open reading frames are created in edited mRNAs and that potentially deleterious modification of normally nonedited sequence does not occur. Selection-amplification and mutagenesis were previously used to identify the optimal sequence requirements for in vitro editing. There is, however, a minority of natural editing sites with suboptimal sequence. Several cis-acting elements, obtained from an in vitro selection, are described here that are able to compensate for a suboptimal editing site. An A + U sequence element within the 5'-untranslated region of cytochrome b mRNA from Leishmania tarentolae is also demonstrated to function as a cis-acting guide RNA and is postulated to compensate for a suboptimal editing site in vivo. Two proteins within an enriched editing extract are UV-cross-linked to two different in vitro selected editing substrates more efficiently than poorly edited RNAs. The results suggest that these proteins contribute to the specificity of the editing reaction.

Animals↗