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In Trypanosoma cruzi DNA is packaged into chromatin by octamers of histone proteins that form nucleosomes. Transcription of protein coding genes in trypanosomes is constitutive producing polycistronic units and gene expression is primarily regulated post-transcriptionally. However, chromatin organization influences DNA dependent processes. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities found in trypanosomes. To map nucleosomes genome-wide in several organisms, digestion of chromatin with micrococcal nuclease followed by deep sequencing has been applied. Nonetheless, the special requirements for cell manipulation and the uniqueness of the chromatin organization in trypanosomes entails a customized analytical approach. In this work, we adjusted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we implemented an exhaustive and thorough computational workflow to overcome the difficulties imposed by this complex genome. We tested the performance of two aligners, Bowtie2 and HISAT2, and discuss their advantages and caveats. Specifically, we highlight the relevance of using the whole genome as a reference instead of the commonly used Esmeraldo-like haplotype to avoid spurious alignments. Additionally, we show that using the whole genome refines the average nucleosome representation, but also the quality of mapping for every region represented. Moreover, we show that the average nucleosome organization around trans-splicing acceptor site described before, is not just an average since the same chromatin pattern is detected for most of the represented regions. In addition, we extended the study to a non-hybrid strain applying the experimental and analytical approach to Sylvio-X10 strain. Furthermore, we provide a source code for the construction of 2D plots and heatmaps which are easy to adapt to any T. cruzi strain.

Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, exhibits peculiar biological features. Among them, the presence of a unique mitochondrion is remarkable. Even though the mitochondrial DNA constitutes up to 25% of total cellular DNA, the structure and functionality of the mitochondrion are dependent on the expression of the nuclear genome. As in other eukaryotes, specific peptide signals have been proposed to drive the mitochondrial localization of a subset of trypanosomatid proteins. However, there are mitochondrial proteins encoded in the nuclear genome that lack of a peptide signal. In other eukaryotes, alternative protein targeting to subcellular organelles via mRNA localization has also been recognized and specific mRNA localization towards the mitochondria has been described. With the aim of seeking for mitochondrial localization signals in T. cruzi, we developed a strategy to build a comprehensive database of nuclear genes encoding predicted mitochondrial proteins (MiNT) in the TriTryps (T. cruzi, T. brucei and L. major). We found that approximately 15% of their nuclear genome encodes mitochondrial products. In T. cruzi the MiNT database reaches 1438 genes and a conserved peptide signal, M(L/F) R (R/S) SS, named TryM-TaPe is found in 60% of these genes, suggesting that the canonical mRNA guidance mechanism is present. In addition, the search for compositional signals in the transcripts of T. cruzi MiNT genes produce a list, being worth to note a conserved non-translated element represented by the consensus sequence DARRVSG. Taking into account its reported interaction with the T. brucei TRRM3 protein which is enriched in the mitochondrial membrane fraction, we here suggest a putative zip code role for this element. Globally, here we provide an inventory of the mitochondrial proteins in T. cruzi and give evidence for the existence of both peptide and mRNA signals specific to nuclear encoded mitochondrial proteins.

Trypanosoma cruzi is the protozoan parasite causing American trypanosomiasis or Chagas disease, a neglected parasitosis with important human health impact in Latin America. The efficacy of current therapy is limited, and its toxicity is high. Since parasite proliferation is a fundamental target for rational drug design, we sought to progress into its understanding by applying a genome-wide approach. Treating a TcI linage strain with hydroxyurea, we isolated epimastigotes in late G1, S and G2/M cell cycle stages at 70% purity. The sequencing of each phase identified 305 stage-specific transcripts (1.5-fold change, p≤0.01), coding for conserved cell cycle regulated proteins and numerous proteins whose cell cycle dependence has not been recognized before. Comparisons with the parasite T. brucei and the human host reveal important differences. The meta-analysis of T. cruzi transcriptomic and ribonomic data indicates that cell cycle regulated mRNAs are subject to sub-cellular compartmentalization. Compositional and structural biases of these genes- including CAI, GC content, UTR length, and polycistron position- may contribute to their regulation. To discover nucleotide motifs responsible for the co-regulation of cell cycle regulated genes, we looked for overrepresented motifs at their UTRs and found a variant of the cell cycle sequence motif at the 3' UTR of most of the S and G2 stage genes. We additionally identified hairpin structures at the 5' UTRs of a high proportion of the transcripts, suggesting that periodic gene expression might also rely on translation initiation in T. cruzi. In summary, we report a comprehensive list of T. cruzi cell cycle regulated genes, including many previously unstudied proteins, we show evidence favoring a multi-step control of their expression, and we identify mRNA motifs that may mediate their regulation. Our results provide novel information of the T. cruzi proliferative proteins and the integrated levels of their gene expression control.

Leishmania major is a flagellated protozoan parasite of medical importance. Like other members of the Trypanosomatidae family, it possesses unique mechanisms of gene expression such as constitutive polycistronic transcription of directional gene clusters, gene amplification, mRNA trans-splicing, and extensive editing of mitochondrial transcripts. The molecular signals underlying most of these processes remain under investigation. In order to investigate the role of DNA secondary structure signals in gene expression, we carried out a genome-wide in silico analysis of the intrinsic DNA curvature. The L. major genome revealed a lower frequency of high intrinsic curvature regions as well as inter- and intra- chromosomal distribution heterogeneity, when compared to prokaryotic and eukaryotic organisms. Using a novel method aimed at detecting region-integrated intrinsic curvature (RIIC), high DNA curvature was found to be associated with regions implicated in transcription initiation. Those include divergent strand-switch regions between directional gene clusters and regions linked to markers of active transcription initiation such as acetylated H3 histone, TRF4 and SNAP50. These findings suggest a role for DNA curvature in transcription initiation in Leishmania supporting the relevance of DNA secondary structures signals.

In Trypanosoma cruzi DNA is packaged into chromatin by octamers of histone proteins that form nucleosomes. Transcription of protein coding genes in trypanosomes is constitutive producing polycistronic units and gene expression is primarily regulated post-transcriptionally. However, chromatin organization influences DNA dependent processes. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities found in trypanosomes. To map nucleosomes genome-wide in several organisms, digestion of chromatin with micrococcal nuclease followed by deep sequencing has been applied. Nonetheless, the special requirements for cell manipulation and the uniqueness of the chromatin organization in trypanosomes entails a customized analytical approach. In this work, we adjusted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we implemented an exhaustive and thorough computational workflow to overcome the difficulties imposed by this complex genome. We tested the performance of two aligners, Bowtie2 and HISAT2, and discuss their advantages and caveats. Specifically, we highlight the relevance of using the whole genome as a reference instead of the commonly used Esmeraldo-like haplotype to avoid spurious alignments. Additionally, we show that using the whole genome refines the average nucleosome representation, but also the quality of mapping for every region represented. Moreover, we show that the average nucleosome organization around trans-splicing acceptor site described before, is not just an average since the same chromatin pattern is detected for most of the represented regions. In addition, we extended the study to a non-hybrid strain applying the experimental and analytical approach to Sylvio-X10 strain. Furthermore, we provide a source code for the construction of 2D plots and heatmaps which are easy to adapt to any T. cruzi strain., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Beati et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, exhibits peculiar biological features. Among them, the presence of a unique mitochondrion is remarkable. Even though the mitochondrial DNA constitutes up to 25% of total cellular DNA, the structure and functionality of the mitochondrion are dependent on the expression of the nuclear genome. As in other eukaryotes, specific peptide signals have been proposed to drive the mitochondrial localization of a subset of trypanosomatid proteins. However, there are mitochondrial proteins encoded in the nuclear genome that lack of a peptide signal. In other eukaryotes, alternative protein targeting to subcellular organelles via mRNA localization has also been recognized and specific mRNA localization towards the mitochondria has been described. With the aim of seeking for mitochondrial localization signals in T. cruzi, we developed a strategy to build a comprehensive database of nuclear genes encoding predicted mitochondrial proteins (MiNT) in the TriTryps (T. cruzi, T. brucei and L. major). We found that approximately 15% of their nuclear genome encodes mitochondrial products. In T. cruzi the MiNT database reaches 1438 genes and a conserved peptide signal, M(L/F) R (R/S) SS, named TryM-TaPe is found in 60% of these genes, suggesting that the canonical mRNA guidance mechanism is present. In addition, the search for compositional signals in the transcripts of T. cruzi MiNT genes produce a list, being worth to note a conserved non-translated element represented by the consensus sequence DARRVSG. Taking into account its reported interaction with the T. brucei TRRM3 protein which is enriched in the mitochondrial membrane fraction, we here suggest a putative zip code role for this element. Globally, here we provide an inventory of the mitochondrial proteins in T. cruzi and give evidence for the existence of both peptide and mRNA signals specific to nuclear encoded mitochondrial proteins., Competing Interests: The authors have declared that no competing interests exist.

Trypanosoma cruzi is the protozoan parasite causing American trypanosomiasis or Chagas disease, a neglected parasitosis with important human health impact in Latin America. The efficacy of current therapy is limited, and its toxicity is high. Since parasite proliferation is a fundamental target for rational drug design, we sought to progress into its understanding by applying a genome-wide approach. Treating a TcI linage strain with hydroxyurea, we isolated epimastigotes in late G1, S and G2/M cell cycle stages at 70% purity. The sequencing of each phase identified 305 stage-specific transcripts (1.5-fold change, p≤0.01), coding for conserved cell cycle regulated proteins and numerous proteins whose cell cycle dependence has not been recognized before. Comparisons with the parasite T. brucei and the human host reveal important differences. The meta-analysis of T. cruzi transcriptomic and ribonomic data indicates that cell cycle regulated mRNAs are subject to sub-cellular compartmentalization. Compositional and structural biases of these genes- including CAI, GC content, UTR length, and polycistron position- may contribute to their regulation. To discover nucleotide motifs responsible for the co-regulation of cell cycle regulated genes, we looked for overrepresented motifs at their UTRs and found a variant of the cell cycle sequence motif at the 3' UTR of most of the S and G2 stage genes. We additionally identified hairpin structures at the 5' UTRs of a high proportion of the transcripts, suggesting that periodic gene expression might also rely on translation initiation in T. cruzi. In summary, we report a comprehensive list of T. cruzi cell cycle regulated genes, including many previously unstudied proteins, we show evidence favoring a multi-step control of their expression, and we identify mRNA motifs that may mediate their regulation. Our results provide novel information of the T. cruzi proliferative proteins and the integrated levels of their gene expression control.

Leishmania major is a flagellated protozoan parasite of medical importance. Like other members of the Trypanosomatidae family, it possesses unique mechanisms of gene expression such as constitutive polycistronic transcription of directional gene clusters, gene amplification, mRNA trans-splicing, and extensive editing of mitochondrial transcripts. The molecular signals underlying most of these processes remain under investigation. In order to investigate the role of DNA secondary structure signals in gene expression, we carried out a genome-wide in silico analysis of the intrinsic DNA curvature. The L. major genome revealed a lower frequency of high intrinsic curvature regions as well as inter- and intra- chromosomal distribution heterogeneity, when compared to prokaryotic and eukaryotic organisms. Using a novel method aimed at detecting region-integrated intrinsic curvature (RIIC), high DNA curvature was found to be associated with regions implicated in transcription initiation. Those include divergent strand-switch regions between directional gene clusters and regions linked to markers of active transcription initiation such as acetylated H3 histone, TRF4 and SNAP50. These findings suggest a role for DNA curvature in transcription initiation in Leishmania supporting the relevance of DNA secondary structures signals.