The RNA editing process in Trypanosoma brucei

Twelve mitochondrial mRNAs are edited in Trypanosoma brucei, nine extensively, by addition and removal of uridines. The accumulation of the edited RNAs is regulated during the life cycle. Hundreds of different gRNAs, encoded three or four per minicircle, specify the editing and minicircle content accounts for variation in editing among species and in mutants. The current understanding of the process of gRNA utilization, the editing mechanism and the editing machinery is discussed.     

Trypanosoma brucei minicircles encode multiple guide RNAs which can direct editing of extensively overlapping sequences.

Small guide RNAs (gRNAs) may direct RNA editing in kinetoplastid mitochondria. We have characterized multiple gRNA genes from Trypanosoma brucei (EATRO 164), that can specify up to 30% of the editing of the COIII, ND7, ND8, and A6 mRNAs and we have also found that the non-translated region of edited COIII mRNA of strain (EATRO 164) differs from that of another strain. Several of the gRNAs specify overlapping regions of the same mRNA often specifying sequence beyond that required for an anchor duplex with the next gRNA. Some gRNAs have different sequence but specify identical editing of the same region of mRNA. These data indicate a complex gRNA population and consequent complex pattern of editing in T. brucei.     

Minicircle-encoded guide RNAs from Crithidia fasciculata.

Although the mitochondrial uridine insertion/deletion, guide RNA (gRNA)-mediated type of RNA editing has been described in Crithidia fasciculata, no evidence for the encoding of gRNAs 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, because 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.     

The mitochondrial tRNAs of Trypanosoma brucei are nuclear encoded

The mitochondrial DNA of Trypanosoma brucei is organized as a catenated network of maxicircles and minicircles. The maxicircles are equivalent to the typical mitochondrial genome except that the genes for the mitochondrial tRNAs have not been identified by sequence analysis of the maxicircle DNA. The apparent absence of tRNA genes in the maxicircle DNA suggests that the mitochondrial tRNAs are encoded by either the minicircle or the nuclear DNA. In order to determine their genomic origin, we isolated and identified the mitochondrial tRNAs of T. brucei. We show that these mitochondrial tRNAs are truly mitochondrially located in vivo and that they are free from detectable contamination by cytosolic RNAs. By hybridization analysis, using mitochondrial tRNAs as the probe, we determined that the mitochondrial tRNAs are encoded by nuclear DNA. This implies that RNAs, like proteins, are imported into the mitochondria. We investigated the relationship between the cytosolic and the mitochondrial tRNA genes and show that there are unique cytosolic tRNA genes, unique mitochondrial tRNA genes, and tRNA genes which appear to be shared and whose products are therefore targeted to both the cytosol and the mitochondrion.     

The insect-phase gRNA transcriptome in Trypanosoma brucei

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.     

Organization and complexity of minicircle-encoded guide RNAs in Trypanosoma cruzi.

The previously observed extensive sequence heterogeneity of the kinetoplast minicircle DNA in Trypanosoma cruzi, both intra- and interstrain, has raised the question as to how the minicircle DNA in this species can have any guide RNA (gRNA)-coding capacity at all, because there do not appear to be any variable-region sequences conserved between different strains. To address this question, we obtained the complete edited sequence of maxicircle unidentified reading frame 4 mRNA and identified 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 nt 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 that encode identical editing information but have different sequences.