In silico screen identifies a new Toxoplasma gondii mitochondrial ribosomal protein essential for mitochondrial translation

SummaryApicomplexan parasites cause diseases such as malaria and toxoplasmosis. The apicomplexan mitochondrion shows striking differences from common model organisms, including in fundamental processes such as mitochondrial translation. Despite evidence that mitochondrial translation is essential for parasites survival, it is largely understudied. Progress has been restricted by the absence of functional assays to detect apicomplexan mitochondrial translation, a lack of knowledge of proteins involved in the process and the inability to identify and detect mitoribosomes.Using mRNA expression patterns, 279 candidate mitochondrial housekeeping components were identified in Toxoplasma. 11 were validated, including the mitoribosomal small subunit protein 35 (TgmS35). TgmS35 tagging enabled the detection of a macromolecular complex corresponding to the mitoribosomal small subunit for the first time in apicomplexans. A new analytical pipeline detected defects in mitochondrial translation upon TgmS35 depletion, while other mitochondrial functions remain unaffected. Our work lays a foundation for the study of apicomplexan mitochondrial translation.Abbreviated summaryThe apicomplexan mitochondrion is divergent and essential yet poorly studied. Mitochondrial translation is predicted to utilize ribosomes assembled from fragmented rRNA but this was never shown. Knowing the mitochondrial protein content is critical for these studies. We identified 11 new mitochondrial proteins via in-silico searches. Tagging and depletion of a mitoribosomal small subunit protein enabled the first detection of a macromolecular ribosomal complex, and provided proof of principle for our new mitochondrial translation analytic pipeline.

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In silico prediction of mtDNA Gene Expression Based on Codon Usage Bias in Ants (Formicidae Latreille, 1802) that Inhabit Limestone Quarry Ecosystems

Genetics & Applications ◽

10.31383/ga.vol2iss1pp32-37 ◽

2018 ◽

Vol 2 (1) ◽

pp. 32

Author(s):

Semir Dorić ◽

Dinko Osmanković ◽

Lada Lukić Bilela

Keyword(s):

Gene Expression ◽

Codon Usage ◽

Codon Usage Bias ◽

In Silico ◽

Expression Patterns ◽

Mitochondrial Protein ◽

Model Organisms ◽

Molecular Response ◽

Protein Coding ◽

Protein Coding Genes

Codon usage is considered as a modulator of gene expression, due to high correlation between codon usage, tRNA abundance and the level of gene expression. Adaptability is primarily manifested at gene level therefore mtDNA gene expression analysis may indicate trends toward the development of adaptive traits for specific environmental conditions. Moreover, modified gene expression patterns may result from such adaptations. Due to their sensitivity to environmental disturbances, great functional importance and accessibility ants (Family: Formicidae Latreille, 1802) are excellent model organisms for molecular and bioinformatics genome analysis. This in silico simulation is based on the comparison of codon usage bias and the level of gene expression of currently available mitochondrial protein-coding genes of ant species that were sampled at quarry Ribnica (Kakanj, Bosnia and Herzegovina). MILC and MELP algorithms were used forcodon usage bias analysis and mitochondrial gene expression prediction, respectively. The analysis included four mtDNA protein-coding genes from eight selected species of ants totaling in 32 protein sequences. The results of codon usage analysis indicated no statistically significant differences in codon usage bias, as well as relative frequencies of the gene expression level. The next step should be directed to molecular ecology studies, even using whole genome measures of gene expression (RNA-seq; transcriptomics) to capture molecular response to environmental challenges.

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Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype

Life ◽

10.3390/life11070674 ◽

2021 ◽

Vol 11 (7) ◽

pp. 674

Author(s):

Francesco Capriglia ◽

Francesca Rizzo ◽

Giuseppe Petrosillo ◽

Veronica Morea ◽

Giulia d’Amati ◽

...

Keyword(s):

Protein Synthesis ◽

Molecular Mechanisms ◽

Mitochondrial Protein ◽

Trna Synthetase ◽

Mitochondrial Protein Synthesis ◽

Mitochondrial Translation ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Mitochondrial Functions ◽

Carboxy Terminal

The m.3243A>G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.

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MRPS25 mutations impair mitochondrial translation and cause encephalomyopathy

Human Molecular Genetics ◽

10.1093/hmg/ddz093 ◽

2019 ◽

Vol 28 (16) ◽

pp. 2711-2719 ◽

Cited By ~ 11

Author(s):

Enrico Bugiardini ◽

Alice L Mitchell ◽

Ilaria Dalla Rosa ◽

Hue-Tran Horning-Do ◽

Alan M Pitmann ◽

...

Keyword(s):

Respiratory Chain ◽

Mitochondrial Protein ◽

In Silico Analysis ◽

Small Subunit ◽

Mitochondrial Translation ◽

Transgenic Expression ◽

Mitochondrial Ribosomes ◽

Mitochondrial Ribosome ◽

Protein Components ◽

Biochemical Analyses

Abstract Mitochondrial disorders are clinically and genetically heterogeneous and are associated with a variety of disease mechanisms. Defects of mitochondrial protein synthesis account for the largest subgroup of disorders manifesting with impaired respiratory chain capacity; yet, only a few have been linked to dysfunction in the protein components of the mitochondrial ribosomes. Here, we report a subject presenting with dyskinetic cerebral palsy and partial agenesis of the corpus callosum, while histochemical and biochemical analyses of skeletal muscle revealed signs of mitochondrial myopathy. Using exome sequencing, we identified a homozygous variant c.215C>T in MRPS25, which encodes for a structural component of the 28S small subunit of the mitochondrial ribosome (mS25). The variant segregated with the disease and substitutes a highly conserved proline residue with leucine (p.P72L) that, based on the high-resolution structure of the 28S ribosome, is predicted to compromise inter-protein contacts and destabilize the small subunit. Concordant with the in silico analysis, patient’s fibroblasts showed decreased levels of MRPS25 and other components of the 28S subunit. Moreover, assembled 28S subunits were scarce in the fibroblasts with mutant mS25 leading to impaired mitochondrial translation and decreased levels of multiple respiratory chain subunits. Crucially, these abnormalities were rescued by transgenic expression of wild-type MRPS25 in the mutant fibroblasts. Collectively, our data demonstrate the pathogenicity of the p.P72L variant and identify MRPS25 mutations as a new cause of mitochondrial translation defect.

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technical knockout, a Drosophila Model of Mitochondrial Deafness

Genetics ◽

10.1093/genetics/159.1.241 ◽

2001 ◽

Vol 159 (1) ◽

pp. 241-254

Author(s):

Janne M Toivonen ◽

Kevin M C O'Dell ◽

Nathalie Petit ◽

Sharon C Irvine ◽

Gillian K Knight ◽

...

Keyword(s):

Mitochondrial Protein ◽

Sensorineural Deafness ◽

Nuclear Gene ◽

Small Subunit ◽

Female Sterility ◽

Small Subunit Rrna ◽

Mitochondrial Translation ◽

Drosophila Model ◽

Ribosomal Protein S12 ◽

Translational Apparatus

Abstract Mutations in mtDNA-encoded components of the mitochondrial translational apparatus are associated with diverse pathological states in humans, notably sensorineural deafness. To develop animal models of such disorders, we have manipulated the nuclear gene for mitochondrial ribosomal protein S12 in Drosophila (technical knockout, tko). The prototypic mutant tko25t exhibits developmental delay, bang sensitivity, impaired male courtship, and defective response to sound. On the basis of a transgenic reversion test, these phenotypes are attributable to a single substitution (L85H) at a conserved residue of the tko protein. The mutant is hypersensitive to doxycyclin, an antibiotic that selectively inhibits mitochondrial protein synthesis, and mutant larvae have greatly diminished activities of mitochondrial redox enzymes and decreased levels of mitochondrial small-subunit rRNA. A second mutation in the tko gene, Q116K, which is predicted to impair the accuracy of mitochondrial translation, results in the completely different phenotype of recessive female sterility, based on three independent transgenic insertions. We infer that the tko25t mutant provides a model of mitochondrial hearing impairment resulting from a quantitative deficiency of mitochondrial translational capacity.

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An unexpectedly complex mitoribosome in Andalucia godoyi, a protist with the most bacteria-like mitochondrial genome

Molecular Biology and Evolution ◽

10.1093/molbev/msaa223 ◽

2020 ◽

Author(s):

Matus Valach ◽

José Angel Gonzalez Alcazar ◽

Matt Sarrasin ◽

B Franz Lang ◽

Michael W Gray ◽

...

Keyword(s):

Large Subunit ◽

Substantial Reduction ◽

Small Subunit ◽

Experimental Information ◽

Model Organisms ◽

Mitochondrial Genomes ◽

Mitochondrial Translation ◽

Evolutionary Trajectories ◽

High Conservation ◽

Genome Analyses

Abstract The mitoribosome, as known from studies in model organisms, deviates considerably from its ancestor, the bacterial ribosome. Deviations include substantial reduction of the mitochondrial ribosomal RNA (mt-rRNA) structure and acquisition of numerous mitochondrion-specific (M) mitoribosomal proteins (mtRPs). A broadly accepted view assumes that M-mtRPs compensate for structural destabilization of mt-rRNA resulting from its evolutionary remodeling. Since most experimental information on mitoribosome makeup comes from eukaryotes having derived mitochondrial genomes and mt-rRNAs, we tested this assumption by investigating the mitochondrial translation machinery of jakobids, a lineage of unicellular protists with the most bacteria-like mitochondrial genomes. We report here proteomics analyses of the Andalucia godoyi small mitoribosomal subunit and in silico transcriptomic and comparative genome analyses of four additional jakobids. Jakobids have mt-rRNA structures that minimally differ from their bacterial counterparts. Yet, with at least 31 small subunit (SSU) and 44 large subunit (LSU) mtRPs, the mitoriboproteome of Andalucia is essentially as complex as that in animals or fungi. Further, the relatively high conservation of jakobid sequences has helped to clarify the identity of several mtRPs, previously considered to be lineage-specific, as divergent homologs of conserved M-mtRPs, notably mS22 and mL61. The coexistence of bacteria-like mt-rRNAs and a complex mitoriboproteome refutes the view that M-mtRPs were ancestrally recruited to stabilize deviations of mt-rRNA structural elements. We postulate instead that the numerous M-mtRPs acquired in the last eukaryotic common ancestor allowed mt-rRNAs to pursue a broad range of evolutionary trajectories across lineages: from dramatic reduction to acquisition of novel elements to structural conservatism.

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In Silico Characterization of Toxin-Antitoxin Systems in Campylobacter Isolates Recovered from Food Sources and Sporadic Human Illness

Genes ◽

10.3390/genes12010072 ◽

2021 ◽

Vol 12 (1) ◽

pp. 72

Author(s):

Bishoy Wadie ◽

Mohamed A. Abdel-Fattah ◽

Alshymaa Yousef ◽

Shaimaa F. Mouftah ◽

Mohamed Elhadidy ◽

...

Keyword(s):

Environmental Conditions ◽

In Silico ◽

Antimicrobial Agents ◽

Food Sources ◽

Model Organisms ◽

Food Borne ◽

Food Borne Illness ◽

Human Illness ◽

Clonal Population Structure ◽

In Silico Characterization

Campylobacter spp. represents the most common cause of gastroenteritis worldwide with the potential to cause serious sequelae. The ability of Campylobacter to survive stressful environmental conditions has been directly linked with food-borne illness. Toxin-antitoxin (TA) modules play an important role as defense systems against antimicrobial agents and are considered an invaluable strategy harnessed by bacterial pathogens to survive in stressful environments. Although TA modules have been extensively studied in model organisms such as Escherichia coli K12, the TA landscape in Campylobacter remains largely unexplored. Therefore, in this study, a comprehensive in silico screen of 111 Campylobacter (90 C.jejuni and 21 C.coli) isolates recovered from different food and clinical sources was performed. We identified 10 type II TA systems belonging to four TA families predicted in Campylobacter genomes. Furthermore, there was a significant association between the clonal population structure and distribution of TA modules; more specifically, most (12/13) of the Campylobacter isolates belonging to ST-21 isolates possess HicB-HicA TA modules. Finally, we observed a high degree of shared synteny among isolates bearing certain TA systems or even coexisting pairs of TA systems. Collectively, these findings provide useful insights about the distribution of TA modules in a heterogeneous pool of Campylobacter isolates from different sources, thus developing a better understanding regarding the mechanisms by which these pathogens survive stressful environmental conditions, which will further aid in the future designing of more targeted antimicrobials.

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Genome-wide characterization of 2-oxoglutarate and Fe(II)-dependent dioxygenase family genes in tomato during growth cycle and their roles in metabolism

BMC Genomics ◽

10.1186/s12864-021-07434-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Shuo Wei ◽

Wen Zhang ◽

Rao Fu ◽

Yang Zhang

Keyword(s):

Metabolic Pathways ◽

Developmental Stages ◽

Expression Patterns ◽

Growth Cycle ◽

Model Organisms ◽

Gibberellin Biosynthesis ◽

Type I ◽

Multiple Model ◽

Genome Wide ◽

Tomato Growth

Abstract Background 2-Oxoglutarate and Fe(II)-dependent dioxygenases (2ODDs) belong to the 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily and are involved in various vital metabolic pathways of plants at different developmental stages. These proteins have been extensively investigated in multiple model organisms. However, these enzymes have not been systematically analyzed in tomato. In addition, type I flavone synthase (FNSI) belongs to the 2ODD family and contributes to the biosynthesis of flavones, but this protein has not been characterized in tomato. Results A total of 131 2ODDs from tomato were identified and divided into seven clades by phylogenetic classification. The Sl2ODDs in the same clade showed similar intron/exon distributions and conserved motifs. The Sl2ODDs were unevenly distributed across the 12 chromosomes, with different expression patterns among major tissues and at different developmental stages of the tomato growth cycle. We characterized several Sl2ODDs and their expression patterns involved in various metabolic pathways, such as gibberellin biosynthesis and catabolism, ethylene biosynthesis, steroidal glycoalkaloid biosynthesis, and flavonoid metabolism. We found that the Sl2ODD expression patterns were consistent with their functions during the tomato growth cycle. These results indicated the significance of Sl2ODDs in tomato growth and metabolism. Based on this genome-wide analysis of Sl2ODDs, we screened six potential FNSI genes using a phylogenetic tree and coexpression analysis. However, none of them exhibited FNSI activity. Conclusions Our study provided a comprehensive understanding of the tomato 2ODD family and demonstrated the significant roles of these family members in plant metabolism. We also suggest that no FNSI genes in tomato contribute to the biosynthesis of flavones.

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Genome-Wide Expression Patterns of Rhoptry Kinases during the Eimeria tenella Life-Cycle

Microorganisms ◽

10.3390/microorganisms9081621 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1621

Author(s):

Adeline Ribeiro E Silva ◽

Alix Sausset ◽

Françoise I. Bussière ◽

Fabrice Laurent ◽

Sonia Lacroix-Lamandé ◽

...

Keyword(s):

Life Cycle ◽

Expression Patterns ◽

Housekeeping Genes ◽

Catalytic Triad ◽

Eimeria Tenella ◽

Apicomplexan Parasites ◽

Genome Wide ◽

Infected Cells ◽

First And Second Generation

Kinome from apicomplexan parasites is composed of eukaryotic protein kinases and Apicomplexa specific kinases, such as rhoptry kinases (ROPK). Ropk is a gene family that is known to play important roles in host–pathogen interaction in Toxoplasma gondii but is still poorly described in Eimeria tenella, the parasite responsible for avian coccidiosis worldwide. In the E. tenella genome, 28 ropk genes are predicted and could be classified as active (n = 7), inactive (incomplete catalytic triad, n = 12), and non-canonical kinases (active kinase with a modified catalytic triad, n = 9). We characterized the ropk gene expression patterns by real-time quantitative RT-PCR, normalized by parasite housekeeping genes, during the E. tenella life-cycle. Analyzed stages were: non-sporulated oocysts, sporulated oocysts, extracellular and intracellular sporozoites, immature and mature schizonts I, first- and second-generation merozoites, and gametes. Transcription of all those predicted ropk was confirmed. The mean intensity of transcription was higher in extracellular stages and 7–9 ropk were specifically transcribed in merozoites in comparison with sporozoites. Transcriptional profiles of intracellular stages were closely related to each other, suggesting a probable common role of ROPKs in hijacking signaling pathways and immune responses in infected cells. These results provide a solid basis for future functional analysis of ROPK from E. tenella.

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