Search

RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55- and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.

Transcripts from many mitochondrial genes in kinetoplastids undergo RNA editing, a posttranscriptional process which inserts and deletes uridines. By assaying for deletion editing in vitro, we found that the editing activity from Trypanosoma brucei mitochondrial lysates (S.D. Seiwert and K.D. Stuart), Science 266:114-117,1994) sediments with a peak of approximately 20S. RNA helicase, terminal uridylyl transferase, RNA ligase, and adenylation activities, which may have a role in editing, cosediment in a broad distribution, with most of each activity at 35 to 40S. Most ATPase 6 (A6) guide RNA and unedited A6 mRNA sediments at 20 to 30S, with some sedimenting further into the gradient, while most edited A6 mRNA sediments at >35S. Several mitochondrial proteins which cross-link specifically with guide RNA upon UV treatment also sediment in glycerol gradients. Notably, a 65-kDa protein sediments primarily at approximately 20S, a 90-kDa protein sediments at 35 to 40S, and a 25-kDa protein is present at <10S. Most ribonucleoprotein complexes that form with gRNA in vitro sediment at 10 to 20S, except for one, which sediments at 30 to 45S. These results suggest that RNA editing takes place within a multicomponent complex. The potential functions of and relationships between the 20S and 35 to 40S complexes are discussed.

Eukaryotic protein-coding genes are generally transcribed by RNA polymerase II (Pol II), which has a lower transcription rate than that of Pol I. We report here the duplication of two LD1 genes into the rRNA locus and their resultant transcription by Pol I. The multigenic LD1 locus is present in a 2.2-Mb chromosome in all stocks of Leishmania spp. and is also present in multicopy 200- to 450-kb linear chromosomes or multicopy circular DNAs in over 15% of stocks examined. Genomic rearrangement in Leishmania donovani LSB-51.1 resulted in duplication of a 3.9-kb segment of LD1 containing two genes (orfF and orfG) and of a 1.3-kb segment from approximately 10 kb downstream into the rRNA gene repeat region of the 1.2-Mb chromosome. Short sequences (12 or 13 bp) common to the 2.2-Mb LD1 and 1.2-Mb rRNA loci suggest that this gene conversion occurred by homologous recombination. Transcription of the duplicated genes is alpha-amanitin resistant, indicating transcription by Pol I, in contrast to the alpha-amanitin-sensitive (Pol II) transcription of the genes in the 2.2-Mb LD1 locus. This results in higher transcript abundance than expected from the gene copy number in LSB-51.1 and in elevated expression of at least the orfF gene product.

Several genes of the Trypanosoma brucei mitochondrial genome (the maxicircle) encode mRNAs that are so extensively altered by RNA editing that the gene cannot be identified by analysis of the DNA sequence. The 322-nucleotide preedited RNA of one of these genes, CR2, is converted into a 647-nucleotide transcript by the addition of 345 uridines and the deletion of 20 genomically encoded uridines. The fully edited transcript has an open reading frame that predicts a 194-amino-acid protein. This protein, which we name ND9 (NADH dehydrogenase subunit 9), has homology to a subunit of NADH dehydrogenase (respiratory complex I). Seven guide RNAs that can specify edited CR2 sequence have been identified. Steady-state levels of unedited ND9 transcripts are greater in bloodstream than in procyclic forms, but edited ND9 mRNA is present in similar abundance in both life cycle stages.

We have cloned the region spanning the putative promoter from two variant surface glycoprotein gene expression sites that are at each end of chromosome M4 of Trypanosoma brucei IsTat 7. Both expression sites contain a retroposon-like sequence (ESR) pseudogene whose 3' end is approximately 30 bp upstream of the putative expression site promoter. The ESRs from both expression sites share considerable sequence homology and are related to LINE-like elements, especially the T. brucei ingi retroposon. Other ESRs are located on large, but not intermediate or mini-, chromosomes in the IsTaR 1 serodeme, and the total copy number is 10 to 20, similar to that estimated for variant surface glycoprotein expression sites. No DNA rearrangements in the vicinity of the ESR and putative expression site promoter were detected following antigenic switches in the IsTaR 1 serodeme. ESR transcripts are present in bloodstream, but not procyclic, forms. Variation in transcript size and sequence between bloodstream variant antigenic types implies that only the ESR from the active expression site is transcribed. This pattern of expression reflects that of sequences downstream of the putative expression site promoter, suggesting that the region of coordinately controlled expression extends upstream of this promoter.

The maxicircle of Trypanosoma brucei encodes components of the mitochondrial oxidative phosphorylation system, as do other mitochondrial DNAs, but maxicircle gene identification is complicated by extensive editing of some transcripts. We found that transcripts from the CR1 region were extensively edited, as are other transcripts from maxicircle regions which exhibit strong G versus C strand bias. Editing added 259 uridines and removed 46 uridines to produce an approximately 574-nucleotide mature mRNA. Partially edited cDNAs and potential guide RNAs were also characterized. Initiation and termination codons were created, and they defined an open reading frame encoding a predicted protein of 145 amino acids. This protein contains two iron-sulfur cysteine motifs and is homologous to a subunit of NADH dehydrogenase and to other electron-carrier proteins. Higher levels of both edited and unedited CR1 transcripts accumulated in bloodstream forms of the parasite than in procyclic forms, suggesting developmental regulation of CR1 gene expression.

We have identified a new variant surface glycoprotein expression site-associated gene (ESAG) in Trypanosoma brucei, the trypanosome leucine repeat (T-LR) gene. Like most other ESAGs, it is expressed in a life cycle stage-specific manner. The N-terminal 20% of the predicted T-LR protein resembles the metal-binding domains of nucleic acid-binding proteins. The remainder is composed of leucine-rich repeats that are characteristic of protein-binding domains found in a variety of other eucaryote proteins. This is the first report of leucine-rich repeats and potential nucleic acid-binding domains on the same protein. The T-LR gene is adjacent to ESAG 4, which has homology to the catalytic domain of adenylate cyclase. This is intriguing, since yeast adenylate cyclase has a leucine-rich repeat regulatory domain. The leucine-rich repeat and putative metal-binding domains suggest a possible regulatory role that may involve adenylate cyclase activity or nucleic acid binding.