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Trypanosoma brucei possesses a highly complex RNA editing system that uses guide RNAs to direct the insertion and deletion of uridines in mitochondrial mRNAs. These changes extensively alter the target mRNAs and can more than double them in length. Recently, analyses showed that several of the edited genes possess the capacity to encode two different protein products. The overlapped reading frames can be accessed through alternative RNA editing that shifts the translated reading frame. In this study, we analyzed the editing patterns of three putative dual-coding genes, ribosomal protein S12 (RPS12), the 5' editing domain of NADH dehydrogenase subunit 7 (ND7 5'), and C-rich region 3 (CR3). We found evidence that alternatively 5'-edited ND7 5' and CR3 transcripts are present in the transcriptome, providing evidence for the use of dual ORFs in these transcripts. Moreover, we found that CR3 has a complex set of editing pathways that vary substantially between cell lines. These findings suggest that alternative editing can work to introduce genetic variation in a system that selects against nucleotide mutations., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Trypanosoma brucei is transmitted between mammalian hosts by the tsetse fly. In the mammal, they are exclusively extracellular, continuously replicating within the bloodstream. During this stage, the mitochondrion lacks a functional electron transport chain (ETC). Successful transition to the fly, requires activation of the ETC and ATP synthesis via oxidative phosphorylation. This life cycle leads to a major problem: in the bloodstream, the mitochondrial genes are not under selection and are subject to genetic drift that endangers their integrity. Exacerbating this, T. brucei undergoes repeated population bottlenecks as they evade the host immune system that would create additional forces of genetic drift. These parasites possess several unique genetic features, including RNA editing of mitochondrial transcripts. RNA editing creates open reading frames by the guided insertion and deletion of U-residues within the mRNA. A major question in the field has been why this metabolically expensive system of RNA editing would evolve and persist. Here, we show that many of the edited mRNAs can alter the choice of start codon and the open reading frame by alternative editing of the 5' end. Analyses of mutational bias indicate that six of the mitochondrial genes may be dual-coding and that RNA editing allows access to both reading frames. We hypothesize that dual-coding genes can protect genetic information by essentially hiding a non-selected gene within one that remains under selection. Thus, the complex RNA editing system found in the mitochondria of trypanosomes provides a unique molecular strategy to combat genetic drift in non-selective conditions.

The mitochondrial genome of Trypanosoma brucei contains many cryptogenes that must be extensively edited following transcription. The RNA editing process is directed by guide RNAs (gRNAs) that encode the information for the specific insertion and deletion of uridylates required to generate translatable mRNAs. We have deep sequenced the gRNA transcriptome from the bloodstream form of the EATRO 164 cell line. Using conventionally accepted fully edited mRNA sequences, ~1 million gRNAs were identified. In contrast, over 3 million reads were identified in our insect stage gRNA transcriptome. A comparison of the two life cycle transcriptomes show an overall ratio of procyclic to bloodstream gRNA reads of 3.5:1. This ratio varies significantly by gene and by gRNA populations within genes. The variation in the abundance of the initiating gRNAs for each gene, however, displays a trend that correlates with the developmental pattern of edited gene expression. A comparison of related major classes from each transcriptome revealed a median value of ten single nucleotide variations per gRNA. Nucleotide variations were much less likely to occur in the consecutive Watson-Crick anchor region, indicating a very strong bias against G:U base pairs in this region. This work indicates that gRNAs are expressed during both life cycle stages, and that differential editing patterns observed for the different mitochondrial mRNA transcripts are not due to the presence or absence of gRNAs. However, the abundance of certain gRNAs may be important in the developmental regulation of RNA editing.

One of the most striking examples of small RNA regulation of gene expression is the process of RNA editing in the mitochondria of trypanosomes. In these parasites, RNA editing involves extensive uridylate insertions and deletions within most of the mitochondrial messenger RNAs (mRNAs). Over 1200 small guide RNAs (gRNAs) are predicted to be responsible for directing the sequence changes that create start and stop codons, correct frameshifts and for many of the mRNAs generate most of the open reading frame. In addition, alternative editing creates the opportunity for unprecedented protein diversity. In Trypanosoma brucei, the vast majority of gRNAs are transcribed from minicircles, which are approximately one kilobase in size, and encode between three and four gRNAs. The large number (5000-10,000) and their concatenated structure make them difficult to sequence. To identify the complete set of gRNAs necessary for mRNA editing in T. brucei, we used Illumina deep sequencing of purified gRNAs from the procyclic stage. We report a near complete set of gRNAs needed to direct the editing of the mRNAs.

Posttranscriptional editing of trypanosome mitochondrial messenger RNA is directed by small guide RNAs (gRNAs). Using crosslinking techniques, we have previously shown that the gRNA base pairs to the mRNA via a 5' anchor, whereas its 3' U-tail interacts with upstream purine-rich mRNA sequences. The incorporation of crosslinking data into RNA folding programs produced similar structure predictions for all gRNA/mRNA pairs examined. This suggests that gRNA/mRNA pairs can form common secondary structure motifs that may be important for recognition by the editing complex. In this study, the structure of CYb mRNA crosslinked to gCYb-558 was examined using solution-probing techniques. The mRNA/gRNA crosslinked molecules are efficient substrates for gRNA-directed cleavage. In addition, when the cleavage assay is performed in the presence or absence of additional UTP, the activities of both the U-specific exonuclease and terminal uridylyl transferase (tutase) can be detected. These results indicate that a partial editing complex can assemble and function on these substrates suggesting that the crosslink captured the molecules in a biologically relevant interaction. The structure probing data directly show that the U-tail protects several mRNA bases predicted to be involved in the U-tail-mRNA duplex. In combination with our previous studies, these new data provide additional support for the predicted secondary structure of interacting gRNA/mRNA pairs.

We report here the characterization of a developmentally regulated mitochondrial RNA polymerase transcript in the parasitic protozoan, Trypanosoma brucei. The 3822 bp protein-coding region of the T. brucei mitochondrial RNA polymerase (TBMTRNAP) gene is predicted to encode a 1274 amino acid polypeptide, the carboxyl-terminal domain of which exhibits 29-37% identity with the mitochondrial RNA polymerases from other organisms in the molecular databases. Interestingly, the TBMTRNAP mRNA is one of several mature mRNA species post-transcriptionally processed from a stable, polycistronic precursor. Alternative polyadenylation of the TBMTRNAP mRNA produces two mature transcripts that differ by 500 nt and that show stage-specific differences in abundance during the T. brucei life cycle. This alternative polyadenylation event appears to be accompanied by the alternative splicing of a high abundance, non-coding downstream transcript of unknown function. Our finding that the TBMTRNAP gene is transcribed into two distinct mRNAs subject to differential regulation during the T. brucei life cycle suggests that mitochondrial differentiation might be achieved in part through the regulated expression of this gene.

The post-transcriptional processing of various mitochondrial transcripts in kinetoplastids, kRNA editing, adds and removes uridines, producing mature messenger RNAs. This editing seems to be directed by 'guide' RNAs (gRNAs) which are complementary to portions of the mature message. The editing mechanism has been proposed to entail transesterification. Detection of chimaeric gRNA-mRNA molecules, intermediates predicted by transesterification, support this model. We report here the in vitro formation of such chimaeras where endogenous gRNAs are covalently linked to added synthetic mRNA. Addition of gel-purified gRNAs to the standard reaction mix increases chimaera formation. This increase is not observed when the gRNA 3'-hydroxyl group is chemically modified, identifying this terminal hydroxyl as the reactive group. These results provide the first experimental evidence for an in vitro RNA editing event and support the involvement of transesterification as a chemical mechanism.

The mitochondrial MURF4 gene of T. brucei has pronounced G versus C strand bias and heterogeneously sized transcripts, characteristic of genes encoding extensively edited transcripts. We find that MURF4 transcripts of T. brucei are extensively edited throughout by the addition and deletion of numerous uridines, creating potential initiation and termination codons and a continuous open reading frame. A potential guide RNA sequence occurs in a minicircle between inverted repeats. The 5' region of L. tarentolae MURF4 transcripts is also extensively edited, with a created initiation codon. The predicted MURF4 amino acid sequences have homology to those of mitochondrial ATP-ase subunit 6 genes from a variety of organisms. In addition, their hydropathic profiles are quite similar to those of other species. We therefore conclude that MURF4 encodes ATPase subunit 6 genes.