Home Economics American Trypanosomiasis Chagas Disease, Second Edition: One Hundred Years of Research
Maxicircles and minicircles: kDNA coding
Maxicircles contain mitochondrial rRNA genes and genes which encode hydrophobic mitochondrial proteins. These proteins are predominantly involved in the process of oxidative phosphorylation. T. cruzi uses this pathway in its transformation to the epimastigote stage in the vector. Other proteins take part in the glycolytic pathway, used by the parasite in its mammal host.
For T. cruzi, the genome of maxicircles has been mounted and annotated for the CL Brener and Esmeraldo strains by the TIGR-SBRI-KI T. cruzi Sequencing Consortium (TSK-TSC). This maxicircle genome is schematized in Fig. 19.3 (reprinted from the original paper of Westenberger et al.14).
The order of the rRNAs and protein genes on the T. cruzi maxicircle is identical with both the T. brucei and L. tarentolae maxicircles.
Figure 19.3 Maxicircle of the Trypanosoma cruzi CL Brener and Esmeraldo reference strains (from Westenberger et al.14). All annotated genes are shown as arrows indicating coding direction. The noncoding regions of both genomes are distinct from one another, with the exception of a duplicated conserved element lying between the repetitive region and the 12S rRNA.
The selective pressure requirement for active gene production is noticeable in the sequence comparison of the maxicircle coding domain. Comparison of maxicircle encoded genes of T. cruzi with T. brucei, T. lewisi, and T. tarantolae demonstrated that whereas nonedited genes (ND5, ND4, COI, COII, ND1, MURF1, MURF2, Cyb) have a similarity of more than 75%, extensively-edited genes (COIII, ATPase6, ND7, ND8, ND9, CR4, CR5, RPS12) have a similarity of less than 50% only at the DNA level. The similarity of translated edited genes rose however to 75% for the majority of comparisons.15
The absence of selective pressure is obvious in the noncoding domain of maxicircles: no similarity was found between T. cruzi with T. brucei and T. tarantolae. Furthermore, almost no homology was evidenced between two sequenced maxicircles of two different lineages of T. cruzi. Variable sequences of maxicircles could also be potentially used for the determination of T. cruzi lineages. However, the variable sequences of maxicircles of each lineage have not yet been published and it has been considered that the number of maxicircles is 100 times lower than the number of minicircles. Nevertheless, sequences of amplicons of maxicircles fragment contributed to determine T. cruzi strains such Cyb as in Chile isolates (Arenas 2011) or more recently COII-NDI fragment for the parasites isolated from the vector Triatoma protracta in California.16
In 1986, Bonne et al. described the presence of four nonencoded uridylate (U) residues in the mRNA of the maxicircle gene encoding subunit 2 of cytochrome c oxidase (cox) of two kinetoplastids, Trypanosoma brucei and Crithidia fasciculate (for review see Benne17).
Now, we know that the DNA of maxicircles encodes 20 genes, corresponding to edited and nonedited genes. However, the majority of primary transcriptions of the maxicircles cannot be directly translated because they often contain many errors relating to the ORF (open reading frame) and should be “published” before the translation. That is why, at first sight, the genome seems to be lacking several genes characteristic of mitochondrial genomes, whereas other genes lack key elements for the translation, such as initiation codons or contiguous ORFs.
Whereas the information contained in a genomic sequence is, in most cases, accurately reproduced in the RNA, in the case of T. cruzi, the transcription undergoes a genuine correction which modifies its sequence, thanks to the process referred to as “RNA edition.” It regroups addition mechanisms, the suppression, and more rarely, the conversion of nucleotides in precise positions of the coding region of maxicircle primary transcriptions (for review see Stuart et al.18 This process may be defined as a programmed alteration of RNA primary structure enabling the production of a functional sequence. Thus, initiation codons are created, or the correction of internal reading is realized, if not, the transcriptions are unrecognizable for the creation of the ORF.14
Since the discovery of mitochondrial RNA editing, we know that this process is the result of a perfect collaboration between genes contained in the maxicircles and the minicircles. The maxicircles provide preedited RNA and the minicircles contribute RNA guides (gRNA). The existence of these gRNA in T. cruzi and their role in the edition of the transcribed RNA maxicircle has been demonstrated by Avila and Simpson19 and Thomas et al.20 This RNA edition must be extremely accurate in order to avoid the insertion or the suppression of a wrong number of uridines, which would falsify the DNA edition.
The consequence of such errors could lead to the synthesis of an untranslatable reading frame, or modify a senseless sequence giving rise to a full reading frame. The key to this precision lies with the RNA guides.21 They possess a complementary sequence of the edited region which determines the precise number of uridines to add, suppress, or convert.
Nevertheless, the level of the variability in the gRNA sequence, without loss of functional information, is impressive, due to the fact that any link with G or residues of U in the RNAm is not affected by the transition mutations in the gRNA.14 But the genes in the maxicircle must maintain a certain degree of fidelity to the gRNA genes in order that the correction be made.2
The gRNA, in their 50 region, present a sequence called “inking sequence,” which pairs with preedited transcriptions, and their 30 region, particular to RNAg, is a poly tail (U) which could be involved in the stabilization of the mRNA—gRNA complex.
The formation of the first mRNA—gRNA complex is crucial to the activation of the edition process. The gRNA associated with the transcription serves as a matrix
for the insertion or suppression of uridines. In some cases, the edition creates a new inking site for a second gRNA. The consecutive action of the gRNA means that the edition is a 3"—50 oriented process which repeats itself until the mRNA is completely edited.
A series of enzymatic reactions triggered by the pairing of the gRNA enables the endonuclease to cleave the RNA messenger at the level of the first wrongly paired base. The 50 fragment thus formed, is maintained close to the 30 fragment via RNA—RNA interactions bringing into play the poly tail (U) of the gRNA22 and proteins. This group is called “editosome.”
Secondly, in the 30 of 50 fragment, takes place the addition, suppression or the conversion of uridines, thanks to 30 terminal uridylyl transferases (TUTases). The newly added uridines pair with the gRNA. The two RNA fragments (50 and 30), which remain together due to complementarity with gRNA, are finally linked by an RNA ligase giving rise to the mature transcription.21 This multisubunit enzymatic RNA editing complex and its function in T. brucei was recently reviewed by Aphasizheva and Aphasizhev.23
The edition process of maxicircle transcriptions contributes to the evolution of the stages of cellular life and the unusual energetic metabolism of T. cruzi in certain stages of life, passing through rich glucose sanguine trypomastigotes to intracellular amastigotes, and to the poorest epimastigotes living in the energy environment of insects’ intestines.
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