Analysis of mRNA processing at whole transcriptome level, transcriptomic profile and genome sequence refinement of Trypanosoma cruzi

Abstract

The genomic sequence of Trypanosoma cruzi, the protozoan causative of Chagas disease was published more than a decade ago. However, due to their complexity, its complete haploid predicted sequence and therefore its genetic repertoire remains unconfirmed. In this work, we have used RNAseq data to improve the previous genome assembly of Sylvio X10 strain and to define the complete transcriptome at trypomastigote stage (mammalian stage). A total of 22,977 transcripts were identified, of which more than half could be considered novel as they did not match previously annotated genes. Moreover, for the first time in T. cruzi, we are providing their relative abundance levels. We have identified that Sylvio X10 trypomastigotes exhibit a predominance of surface protein genes, specifically those encoding trans-sialidase and mucin-like proteins. On the other hand, detailed analysis of the pre-mRNA processing sites revealed some similarities but also some differences in the spliced leader and different polyadenylation addition sites compared to close related kinetoplastid parasites. Our results also confirm that transcription is bidirectional as occur in other kinetoplastids and the proportion of forward-sense and reverse-sense transcripts is almost equivalent, demonstrating that a strand-specificity does not exist.

Trypanosoma Cruzi Genome: Organization, Multi-Gene Families, Transcription, and Biological Implications

“…be the most probable signal of polyadenylation start, being cytosine the most frequent nucleotide (45.3%) and thymine the less frequent (6.79%) [57]. This differs from other trypanosomatids species, as Leishmania major and T. brucei that presents a AA dinucleotide [97,98] as the most probable signal for polyadenylation.…”

“…Moreover, it was demonstrated that transcriptomic data may be useful to correct and re-annotate previous assembled genomes. Besides, in the case of Sylvio X10/1, RNAseq data was used to improve the previous genome annotation showing that 79.95% of the genome corresponds to the coding sequence, while the previous genomic analysis established only a 37.73% [ 57 ]. These results also suggested that the haploid genome for Sylvio X10/1 may be higher than previously reported (at least 51 Mb).…”

“…Polycistronic transcripts are processed to produce mature mRNAs [ 88 ]. T. cruzi gene clusters can range from 30 to 500 kb separated by divergent or convergent strand-switch regions, or in a head-to-tail orientation whereby transcription terminates and then restarts from the same strand [ 57 , 89 ].…”

“…The AG dinucleotide was described as the consensus sequence for the SL trans-splicing in T. cruzi [ 57 ], Leishmania major [ 97 ] and T. brucei [ 98 ]. However, small differences were detected between all of them in the nucleotide composition surrounding the AG dinucleotide, suggesting that different specific mechanisms are involved in the mRNA maturation among these species.…”

“…The AAUAAA polyadenylation signal of eukaryotes is not present in trypanosomatids. Recent studies published by our group demonstrated that T. cruzi shows a single nucleotide that seems to be the most probable signal of polyadenylation start, being cytosine the most frequent nucleotide (45.3%) and thymine the less frequent (6.79%) [ 57 ]. This differs from other trypanosomatids species, as Leishmania major and T. brucei that presents a AA dinucleotide [ 97 , 98 ] as the most probable signal for polyadenylation.…”