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Kinetoplastids, a group of flagellated protists that are often insect intestinal parasites, encounter various sources of oxidative stress. Such stressors include reactive oxygen species, both internally produced within the protist, and induced externally by host immune responses. This investigation focuses on the role of a highly conserved aspartate-based protein phosphatase, PTP-Interacting protein (PIP39) in managing oxidative stress. In addition to its well accepted role in a Trypanosoma brucei life stage transition, there is evidence of PIP39 participation in the T. brucei oxidative stress response. To examine whether this latter PIP39 role may exist more broadly, we aimed to elucidate PIP39's contribution to redox homeostasis in the monoxenous parasite Leptomonas seymouri. Utilizing CRISPR-Cas9-mediated elimination of PIP39 in conjunction with oxidative stress assays, we demonstrate that PIP39 is required for cellular tolerance to oxidative stress in L. seymouri, positing it as a putative regulatory node for adaptive stress responses. We propose that future analysis of L. seymouri PIP39 enzymatic activity, regulation, and potential localization to a specialized organelle termed a glycosome will contribute to a deeper understanding of the molecular mechanisms by which protozoan parasites adapt to oxidative environments. Our study also demonstrates success at using gene editing tools developed for Leishmania for the related L. seymouri.
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Estrés Oxidativo , Proteínas Protozoarias , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Sistemas CRISPR-Cas , Kinetoplastida/genética , Kinetoplastida/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/genética , Especies Reactivas de Oxígeno/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/fisiologíaRESUMEN
The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.
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Núcleo Celular , Genoma Mitocondrial , Edición de ARN , ARN de Transferencia , Núcleo Celular/genética , Núcleo Celular/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Trypanosomatina/genética , Trypanosomatina/metabolismo , Codón/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Codón de Terminación/genética , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Código Genético , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Instability is an intriguing characteristic of many protist genomes, and trypanosomatids are not an exception in this respect. Some regions of trypanosomatid genomes evolve fast. For instance, the trypanosomatid mitochondrial (kinetoplast) genome consists of fairly conserved maxicircle and minicircle molecules that can, nevertheless, possess high nucleotide substitution rates between closely related strains. Recent experiments have demonstrated that rapid laboratory evolution can result in the non-functionality of multiple genes of kinetoplast genomes due to the accumulation of mutations or loss of critical genomic components. An example of a loss of critical components is the reported loss of entire minicircle classes in Leishmania tarentolae during laboratory cultivation, which results in an inability to generate some correctly encoded genes. In the current work, we estimated the evolutionary rates of mitochondrial and nuclear genome regions of multiple natural Leishmania spp. We analyzed synonymous and non-synonymous substitutions and, rather unexpectedly, found that the coding regions of kinetoplast maxicircles are among the most variable regions of both genomes. In addition, we demonstrate that synonymous substitutions greatly predominate among maxicircle coding regions and that most maxicircle genes show signs of purifying selection. These results imply that maxicircles in natural Leishmania populations remain functional despite their high mutation rate.
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BACKGROUND: Protists of the family Trypanosomatidae (phylum Euglenozoa) have gained notoriety as parasites affecting humans, domestic animals, and agricultural plants. However, the true extent of the group's diversity spreads far beyond the medically and veterinary relevant species. We address several knowledge gaps in trypanosomatid research by undertaking sequencing, assembly, and analysis of genomes from previously overlooked representatives of this protistan group. RESULTS: We assembled genomes for twenty-one trypanosomatid species, with a primary focus on insect parasites and Trypanosoma spp. parasitizing non-human hosts. The assemblies exhibit sizes consistent with previously sequenced trypanosomatid genomes, ranging from approximately 18 Mb for Obscuromonas modryi to 35 Mb for Crithidia brevicula and Zelonia costaricensis. Despite being the smallest, the genome of O. modryi has the highest content of repetitive elements, contributing nearly half of its total size. Conversely, the highest proportion of unique DNA is found in the genomes of Wallacemonas spp., with repeats accounting for less than 8% of the assembly length. The majority of examined species exhibit varying degrees of aneuploidy, with trisomy being the most frequently observed condition after disomy. CONCLUSIONS: The genome of Obscuromonas modryi represents a very unusual, if not unique, example of evolution driven by two antidromous forces: i) increasing dependence on the host leading to genomic shrinkage and ii) expansion of repeats causing genome enlargement. The observed variation in somy within and between trypanosomatid genera suggests that these flagellates are largely predisposed to aneuploidy and, apparently, exploit it to gain a fitness advantage. High heterogeneity in the genome size, repeat content, and variation in chromosome copy numbers in the newly-sequenced species highlight the remarkable genome plasticity exhibited by trypanosomatid flagellates. These new genome assemblies are a robust foundation for future research on the genetic basis of life cycle changes and adaptation to different hosts in the family Trypanosomatidae.
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Trypanosomatina , Animales , Trypanosomatina/genética , Tamaño del Genoma , Aclimatación , Agricultura , AneuploidiaRESUMEN
Since the first identification of circular RNA (circRNA) in viral-like systems, reports of circRNAs and their functions in various organisms, cell types, and organelles have greatly expanded. Here, we report the first evidence, to our knowledge, of circular mRNA in the mitochondrion of the eukaryotic parasite, Trypanosoma brucei. While using a circular RT-PCR technique developed to sequence mRNA tails of mitochondrial transcripts, we found that some mRNAs are circularized without an in vitro circularization step normally required to produce PCR products. Starting from total in vitro circularized RNA and in vivo circRNA, we high-throughput sequenced three transcripts from the 3' end of the coding region, through the 3' tail, to the 5' start of the coding region. We found that fewer reads in the circRNA libraries contained tails than in the total RNA libraries. When tails were present on circRNAs, they were shorter and less adenine-rich than the total population of RNA tails of the same transcript. Additionally, using hidden Markov modelling we determined that enzymatic activity during tail addition is different for circRNAs than for total RNA. Lastly, circRNA UTRs tended to be shorter and more variable than those of the same transcript sequenced from total RNA. We propose a revised model of Trypanosome mitochondrial tail addition, in which a fraction of mRNAs is circularized prior to the addition of adenine-rich tails and may act as a new regulatory molecule or in a degradation pathway.
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MicroARNs , Trypanosoma brucei brucei , ARN Circular/genética , ARN Circular/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN/metabolismo , MicroARNs/metabolismoRESUMEN
Since the first identification of circular RNA (circRNA) in viral-like systems, reports of circRNAs and their functions in various organisms, cell types, and organelles have greatly expanded. Here, we report the first evidence of circular mRNA in the mitochondrion of the eukaryotic parasite, Trypanosoma brucei . While using a circular RT-PCR technique developed to sequence mRNA tails of mitochondrial transcripts, we found that some mRNAs are circularized without an in vitro circularization step normally required to produce PCR products. Starting from total in vitro circularized RNA and in vivo circRNA, we high-throughput sequenced three transcripts from the 3' end of the coding region, through the 3' tail, to the 5' start of the coding region. We found that fewer reads in the circRNA libraries contained tails than in the total RNA libraries. When tails were present on circRNAs, they were shorter and less adenine-rich than the total population of RNA tails of the same transcript. Additionally, using hidden Markov modelling we determined that enzymatic activity during tail addition is different for circRNAs than for total RNA. Lastly, circRNA UTRs tended to be shorter and more variable than those of the same transcript sequenced from total RNA. We propose a revised model of Trypanosome mitochondrial tail addition, in which a fraction of mRNAs is circularized prior to the addition of adenine-rich tails and may act as a new regulatory molecule or in a degradation pathway.
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The kinetoplastids are unicellular flagellates that derive their name from the 'kinetoplast', a region within their single mitochondrion harboring its organellar genome of high DNA content, called kinetoplast (k) DNA. Some protein products of this mitochondrial genome are encoded as cryptogenes; their transcripts require editing to generate an open reading frame. This happens through RNA editing, whereby small regulatory guide (g)RNAs direct the proper insertion and deletion of one or more uridines at each editing site within specific transcript regions. An accurate perspective of the kDNA expansion and evolution of their unique uridine insertion/deletion editing across kinetoplastids has been difficult to achieve. Here, we resolved the kDNA structure and editing patterns in the early-branching kinetoplastid Trypanoplasma borreli and compare them with those of the well-studied trypanosomatids. We find that its kDNA consists of circular molecules of about 42 kb that harbor the rRNA and protein-coding genes, and 17 different contigs of approximately 70 kb carrying an average of 23 putative gRNA loci per contig. These contigs may be linear molecules, as they contain repetitive termini. Our analysis uncovered a putative gRNA population with unique length and sequence parameters that is massive relative to the editing needs of this parasite. We validated or determined the sequence identity of four edited mRNAs, including one coding for ATP synthase 6 that was previously thought to be missing. We utilized computational methods to show that the T. borreli transcriptome includes a substantial number of transcripts with inconsistent editing patterns, apparently products of non-canonical editing. This species utilizes the most extensive uridine deletion compared to other studied kinetoplastids to enforce amino acid conservation of cryptogene products, although insertions still remain more frequent. Finally, in three tested mitochondrial transcriptomes of kinetoplastids, uridine deletions are more common in the raw mitochondrial reads than aligned to the fully edited, translationally competent mRNAs. We conclude that the organization of kDNA across known kinetoplastids represents variations on partitioned coding and repetitive regions of circular molecules encoding mRNAs and rRNAs, while gRNA loci are positioned on a highly unstable population of molecules that differ in relative abundance across strains. Likewise, while all kinetoplastids possess conserved machinery performing RNA editing of the uridine insertion/deletion type, its output parameters are species-specific.
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Trypanosoma cruzi is a unicellular protistan parasitic species that is comprised of strains and isolates exhibiting high levels of genetic and metabolic variability. In the insect vector, it is known to be highly responsive to starvation, a signal for progression to a life stage in which it can infect mammalian cells. Most mRNAs encoded in its mitochondrion require the targeted insertion and deletion of uridines to become translatable transcripts. This study defined differences in uridine-insertion/deletion RNA editing among three strains and established the mechanism whereby abundances of edited (and, thus, translatable) mitochondrial gene products increase during starvation. Our approach utilized our custom T-Aligner toolkit to describe transcriptome-wide editing events and reconstruct editing products from high-throughput sequencing data. We found that the relative abundance of mitochondrial transcripts and the proportion of mRNAs that are edited varies greatly between analyzed strains, a characteristic that could potentially impact metabolic capacity. Starvation typically led to an increase in overall editing activity rather than affecting a specific step in the process. We also determined that transcripts CR3, CR4, and ND3 produce multiple open reading frames that, if translated, would generate different proteins. Finally, we quantitated the inherent flexibility of editing in T. cruzi and found it to be higher relative to that in a related trypanosomatid lineage. Over time, new editing domains or patterns could prove advantageous to the organism and become more widespread within individual transcriptomes or among strains.
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Trypanosoma brucei brucei , Trypanosoma cruzi , Animales , Mamíferos/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN/metabolismo , Edición de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Transcriptoma , Trypanosoma brucei brucei/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMEN
Many medical schools are instituting gender equity initiatives to address long-standing inequities (e.g., salary, leadership positions, resource distribution) between women and men in academic medicine. However, few theory-driven models exist with built-in metrics to assess the impact of gender equity initiatives. The authors describe the theory- and metric-driven process used to create the Center for Women in Medicine and Science (CWIMS) at the University of Minnesota (UMN) Medical School. An innovative theory-driven approach using community-based participatory research (CBPR) was used to create and organize CWIMS. CBPR acknowledges community members (e.g., faculty members, staff), academic organizational representatives (e.g., department heads, center directors), and administrative leaders (e.g., deans) as equal contributors in carrying out all aspects of gender equity work. CBPR values collaborative approaches that empower faculty, promote co-learning and co-creation of initiatives among all university partners, and build upon already existing community strengths and resources. Four CWIMS action groups were created using CBPR principles. The action groups are retention and recruitment; mentoring; salary, resource, and leadership equity; and strategic communications and collaborations. Faculty members across all medical school departments joined these 4 action groups to co-create and carry out all CWIMS gender equity initiatives. The process of developing the CWIMS center and action groups, the CBPR theoretical model guiding the approach, the initiatives developed by the action groups and metrics created, and the outcomes achieved to date are described. In addition, 4 lessons learned from the development of the CWIMS-use of theoretically driven and evidence-based models is key to building a sustainable organization; bottom-up and top-down engagement of partners is crucial for sustainability; passion and innovation are critical for long-term momentum; and not all faculty members and leaders will be enthusiastic about gender equity issues-are shared for the benefit of other medical schools wanting to develop similar centers.
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Equidad de Género , Medicina , Investigación Participativa Basada en la Comunidad , Docentes Médicos , Femenino , Humanos , Liderazgo , Masculino , Facultades de MedicinaRESUMEN
Information, energy, and matter are fundamental properties of all levels of biological organization, and life emerges from the continuous flux of matter, energy, and information. This perspective piece defines and explains each of the three pillars of this nexus. We propose that a quantitative characterization of the complex interconversions between matter, energy, and information that comprise this nexus will help us derive biological insights that connect phenomena across different levels of biological organization. We articulate examples from multiple biological scales that highlight how this nexus approach leads to a more complete understanding of the biological system. Metrics of energy, information, and matter can provide a common currency that helps link phenomena across levels of biological organization. The propagation of energy and information through levels of biological organization can result in emergent properties and system-wide changes that impact other hierarchical levels. Deeper consideration of measured imbalances in energy, information, and matter can help researchers identify key factors that influence system function at one scale, highlighting avenues to link phenomena across levels of biological organization and develop predictive models of biological systems.
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Biología , AnimalesRESUMEN
Uridine insertion/deletion (U-indel) editing of mitochondrial mRNA, unique to the protistan class Kinetoplastea, generates canonical as well as potentially non-productive editing events. While the molecular machinery and the role of the guide (g) RNAs that provide required information for U-indel editing are well understood, little is known about the forces underlying its apparently error-prone nature. Analysis of a gRNA:mRNA pair allows the dissection of editing events in a given position of a given mitochondrial transcript. A complete gRNA dataset, paired with a fully characterized mRNA population that includes non-canonically edited transcripts, would allow such an analysis to be performed globally across the mitochondrial transcriptome. To achieve this, we have assembled 67 minicircles of the insect parasite Leptomonas pyrrhocoris, with each minicircle typically encoding one gRNA located in one of two similar-sized units of different origin. From this relatively narrow set of annotated gRNAs, we have dissected all identified mitochondrial editing events in L. pyrrhocoris, the strains of which dramatically differ in the abundance of individual minicircle classes. Our results support a model in which a multitude of editing events are driven by a limited set of gRNAs, with individual gRNAs possessing an inherent ability to guide canonical and non-canonical editing.
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Genoma de Protozoos , Edición de ARN , ARN Mensajero/metabolismo , ARN Mitocondrial/metabolismo , Trypanosomatina/genética , Filogenia , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/metabolismo , Transcriptoma , Trypanosomatina/metabolismoRESUMEN
A recently redescribed two-flagellar trypanosomatid Vickermania ingenoplastis is insensitive to the classical inhibitors of respiration and thrives under anaerobic conditions. Using genomic and transcriptomic data, we analyzed its genes of the core metabolism and documented that subunits of the mitochondrial respiratory complexes III and IV are ablated, while those of complexes I, II, and V are all present, along with an alternative oxidase. This explains the previously reported conversion of glucose to acetate and succinate by aerobic fermentation. Glycolytic pyruvate is metabolized to acetate and ethanol by pyruvate dismutation, whereby a unique type of alcohol dehydrogenase (shared only with Phytomonas spp.) processes an excess of reducing equivalents formed under anaerobic conditions, leading to the formation of ethanol. Succinate (formed to maintain the glycosomal redox balance) is converted to propionate by a cyclic process involving three enzymes of the mitochondrial methyl-malonyl-CoA pathway, via a cyclic process, which results in the formation of additional ATP. The unusual structure of the V. ingenoplastis genome and its similarity with that of Phytomonas spp. imply their relatedness or convergent evolution. Nevertheless, a critical difference between these two trypanosomatids is that the former has significantly increased its genome size by gene duplications, while the latter streamlined its genome.
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In this study, hierarchies of probabilistic models are evaluated for their ability to characterize the untemplated addition of adenine and uracil to the 3' ends of mitochondrial mRNAs of the human pathogen Trypanosoma brucei, and for their generative abilities to reproduce populations of these untemplated adenine/uridine "tails". We determined the most ideal Hidden Markov Models (HMMs) for this biological system. While our HMMs were not able to generatively reproduce the length distribution of the tails, they fared better in reproducing nucleotide composition aspects of the tail populations. The HMMs robustly identified distinct states of nucleotide addition that correlate to experimentally verified tail nucleotide composition differences. However they also identified a surprising subclass of tails among the ND1 gene transcript populations that is unexpected given the current idea of sequential enzymatic action of untemplated tail addition in this system. Therefore, these models can not only be utilized to reflect biological states that we already know about, they can also identify hypotheses to be experimentally tested. Finally, our HMMs supplied a way to correct a portion of the sequencing errors present in our data. Importantly, these models constitute rare simple pedagogical examples of applied bioinformatic HMMs, due to their binary emissions.
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Enzimas/metabolismo , Modelos Estadísticos , Polimerizacion , Cadenas de Markov , ARN Mensajero/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
In kinetoplastids, the first seven steps of glycolysis are compartmentalized into a glycosome along with parts of other metabolic pathways. This organelle shares a common ancestor with the better-understood eukaryotic peroxisome. Much of our understanding of the emergence, evolution, and maintenance of glycosomes is limited to explorations of the dixenous parasites, including the enzymatic contents of the organelle. Our objective was to determine the extent that we could leverage existing studies in model kinetoplastids to determine the composition of glycosomes in species lacking evidence of experimental localization. These include diverse monoxenous species and dixenous species with very different hosts. For many of these, genome or transcriptome sequences are available. Our approach initiated with a meta-analysis of existing studies to generate a subset of enzymes with highest evidence of glycosome localization. From this dataset we extracted the best possible glycosome signal peptide identification scheme for in silico identification of glycosomal proteins from any kinetoplastid species. Validation suggested that a high glycosome localization score from our algorithm would be indicative of a glycosomal protein. We found that while metabolic pathways were consistently represented across kinetoplastids, individual proteins within those pathways may not universally exhibit evidence of glycosome localization.
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The protozoan Trypanosoma cruzi has a complicated dual-host life cycle, and starvation can trigger transition from the replicating insect stage to the mammalian-infectious nonreplicating insect stage (epimastigote to trypomastigote differentiation). Abundance of some mature RNAs derived from its mitochondrial genome increase during culture starvation of T. cruzi for unknown reasons. Here, we examine T. cruzi mitochondrial gene expression in the mammalian intracellular replicating life stage (amastigote), and uncover implications of starvation-induced changes in gene expression. Mitochondrial RNA levels in general were found to be lowest in actively replicating amastigotes. We discovered that mitochondrial respiration decreases during starvation in insect stage cells, despite the previously observed increases in mitochondrial mRNAs encoding electron transport chain (ETC) components. Surprisingly, T. cruzi epimastigotes in replete medium grow at normal rates when we genetically compromised their ability to perform insertion/deletion editing and thereby generate mature forms of some mitochondrial mRNAs. However, these cells, when starved, were impeded in the epimastigote to trypomastigote transition. Further, they experience a short-flagella phenotype that may also be linked to differentiation. We hypothesize a scenario where levels of mature RNA species or editing in the single T. cruzi mitochondrion are linked to differentiation by a yet-unknown signaling mechanism.
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Regulación de la Expresión Génica , Genes Mitocondriales , Estadios del Ciclo de Vida , ARN Mensajero/genética , ARN Mitocondrial/genética , Trypanosoma cruzi/genética , Células 3T3-L1 , Animales , Diferenciación Celular , Línea Celular , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Humanos , Ratones , Proteínas Protozoarias/metabolismo , Ribosomas/metabolismo , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
What do the products of a genome do, and when and why are they needed? For the protein products of the trypanosomatid parasites' mitochondrial genomes, the total expressed protein repertoire and the identities of the more difficult-to-characterize products have been challenging to acquire. Comparative genomics and new technologies may resolve that.
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Genoma Mitocondrial/genética , Genoma de Protozoos/genética , Trypanosoma/genética , Proteínas Protozoarias/genéticaRESUMEN
Among Euglenozoans, mitochondrial RNA editing occurs in the diplonemids and in the kinetoplastids that include parasitic trypanosomes. Yet U-indel editing, in which open reading frames (ORFs) on mRNAs are generated by insertion and deletion of uridylates in locations dictated by guide RNAs, appears confined to kinetoplastids. The nature of guide RNA and edited mRNA populations has been cursorily explored in a surprisingly extensive number of species over the years, although complete sets of fully edited mRNAs for most kinetoplast genomes are largely missing. Now, however, high throughput sequencing technologies have had an enormous impact on what we know and will learn about the mechanisms, benefits, and final edited products of U-indel editing. Tools including PARERS, TREAT, and T-Aligner function to organize and make sense of U-indel mRNA transcriptomes, which are comprised of mRNAs harboring uridylate indels both consistent and inconsistent with translatable products. From high throughput sequencing data come arguments that partially edited mRNAs containing "junction regions" of noncanonical editing are editing intermediates, and conversely, arguments that they are dead-end products. These data have also revealed that the percent of a given transcript population that is fully or partially edited varies dramatically between transcripts and organisms. Outstanding questions that are being addressed include the prevalence of sequences that apparently encode alternative ORFs, diversity of editing events in ORF termini and 5' and 3' untranslated regions, and the differences that exist in this byzantine process between species. High throughput sequencing technologies will also undoubtedly be harnessed to probe U-indel editing's evolutionary origins. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Evolution and Genomics > Computational Analyses of RNA.
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The insect-transmitted protozoan parasite Trypanosoma cruzi experiences changes in nutrient availability and rate of flux through different metabolic pathways across its life cycle. The species encompasses much genetic diversity of both the nuclear and mitochondrial genomes among isolated strains. The genetic or expression variation of both genomes are likely to impact metabolic responses to environmental stimuli, and even steady state metabolic function, among strains. To begin formal characterization these differences, we compared aspects of metabolism between genetically similar strains CL Brener and Tulahuen with less similar Esmeraldo and Sylvio X10 strains in a culture environment. Epimastigotes of all strains took up glucose at similar rates. However, the degree of medium acidification that could be observed when glucose was absent from the medium varied by strain, indicating potential differences in excreted metabolic byproducts. Our main focus was differences related to electron transport chain function. We observed differences in ATP-coupled respiration and maximal respiratory capacity, mitochondrial membrane potential, and mitochondrial morphology between strains, despite the fact that abundances of two nuclear-encoded proteins of the electron transport chain are similar between strains. RNA sequencing reveals strain-specific differences in abundances of mRNAs encoding proteins of the respiratory chain but also other metabolic processes. From these differences in metabolism and mitochondrial phenotypes we have generated tentative models for the differential metabolic fluxes or differences in gene expression that may underlie these results.
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Regulación de la Expresión Génica , Mitocondrias/metabolismo , Trypanosoma cruzi/citología , Trypanosoma cruzi/crecimiento & desarrollo , Espacio Extracelular/metabolismo , Glucosa/deficiencia , Cinética , Potencial de la Membrana Mitocondrial , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad de la Especie , Termodinámica , Trypanosoma cruzi/genéticaRESUMEN
RNA editing by targeted insertion and deletion of uridine is crucial to generate translatable mRNAs from the cryptogenes of the mitochondrial genome of kinetoplastids. This type of editing consists of a stepwise cascade of reactions generally proceeding from 3' to 5' on a transcript, resulting in a population of partially edited as well as pre-edited and completely edited molecules for each mitochondrial cryptogene of these protozoans. Often, the number of uridines inserted and deleted exceed the number of nucleotides that are genome-encoded. Thus, analysis of kinetoplastid mitochondrial transcriptomes has proven frustratingly complex. Here we present our analysis of Leptomonas pyrrhocoris mitochondrial cDNA deep sequencing reads using T-Aligner, our new tool which allows comprehensive characterization of RNA editing, not relying on targeted transcript amplification and on prior knowledge of final edited products. T-Aligner implements a pipeline of read mapping, visualization of all editing states and their coverage, and assembly of canonical and alternative translatable mRNAs. We also assess T-Aligner functionality on a more challenging deep sequencing read input from Trypanosoma cruzi. The analysis reveals that transcripts of cryptogenes of both species undergo very complex editing that includes the formation of alternative open reading frames and whole categories of truncated editing products.