Cell lineage-specific mitochondrial resilience during mammalian organogenesis.

Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments.

Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs.

Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.

Metabolic shift underlies recovery in reversible infantile respiratory chain deficiency.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.

YbeY is required for ribosome small subunit assembly and tRNA processing in human mitochondria.

Mitochondria contain their own translation apparatus which enables them to produce the polypeptides encoded in their genome. The mitochondrially-encoded RNA components of the mitochondrial ribosome require various post-transcriptional processing steps. Additional protein factors are required to facilitate the biogenesis of the functional mitoribosome. We have characterized a mitochondrially-localized protein, YbeY, which interacts with the assembling mitoribosome through the small subunit. Loss of YbeY leads to a severe reduction in mitochondrial translation and a loss of cell viability, associated with less accurate mitochondrial tRNASer(AGY) processing from the primary transcript and a defect in the maturation of the mitoribosomal small subunit. Our results suggest that YbeY performs a dual, likely independent, function in mitochondria being involved in precursor RNA processing and mitoribosome biogenesis. Issue Section: Nucleic Acid Enzymes.

Regulation of Mammalian Mitochondrial Gene Expression: Recent Advances.

Perturbation of mitochondrial DNA (mtDNA) gene expression can lead to human pathologies. Therefore, a greater appreciation of the basic mechanisms of mitochondrial gene expression is desirable to understand the pathophysiology of associated disorders. Although the purpose of the mitochondrial gene expression machinery is to provide only 13 proteins of the oxidative phosphorylation (OxPhos) system, recent studies have revealed its remarkable and unexpected complexity. We review here the latest breakthroughs in our understanding of the post-transcriptional processes of mitochondrial gene expression, focusing on advances in analyzing the mitochondrial epitranscriptome, the role of mitochondrial RNA granules (MRGs), the benefits of recently obtained structures of the mitochondrial ribosome, and the coordination of mitochondrial and cytosolic translation to orchestrate the biogenesis of OxPhos complexes.

Variants in PUS7 Cause Intellectual Disability with Speech Delay, Microcephaly, Short Stature, and Aggressive Behavior.

We describe six persons from three families with three homozygous protein truncating variants in PUS7: c.89_90del (p.Thr30Lysfs∗20), c.1348C>T (p.Arg450∗), and a deletion of the penultimate exon 15. All these individuals have intellectual disability with speech delay, short stature, microcephaly, and aggressive behavior. PUS7 encodes the RNA-independent pseudouridylate synthase 7. Pseudouridylation is the most abundant post-transcriptional modification in RNA, which is primarily thought to stabilize secondary structures of RNA. We show that the disease-related variants lead to abolishment of PUS7 activity on both tRNA and mRNA substrates. Moreover, pus7 knockout in Drosophila melanogaster results in a number of behavioral defects, including increased activity, disorientation, and aggressiveness supporting that neurological defects are caused by PUS7 variants. Our findings demonstrate that RNA pseudouridylation by PUS7 is essential for proper neuronal development and function.

Outcomes of rotational atherectomy in complex lesions of the superficial femoral artery.

BACKGROUND: The effectiveness of rotational atherectomy in the treatment of complex superficial femoral artery (SFA) lesions remains poorly defined. Outcomes of SFA lesions treated with rotational atherectomy were analyzed. METHODS: This retrospective review assessed all patients who underwent rotational atherectomy of the SFA at a single institution between 2015 and 2018. The data of all patients were deidentified, and the study was approved by the Institutional Review Board. Informed consent was not obtained for this retrospective analysis. Main outcomes were Kaplan-Meier primary patency rate, freedom from major amputation, and 2-year survival rate. The effect of drug-coated balloon angioplasty (DCBA) on patency and time to death was investigated with univariate regression. The safety profile for atherectomy and DCBA was assessed by the 30-day incidence of major amputation and all-cause mortality. RESULTS: Fifty-three patients (mean age, 70.2 ± 9.8 years; 73% male; 65% critical limb-threatening ischemia; 47 [90%] current or former smokers; seven [13%] with prior failed ipsilateral endovascular intervention) underwent rotational atherectomy (Jetstream; Boston Scientific, Marlborough, Mass) with mean follow-up of 543 days. Forty-six (87%) patients underwent DCBA (Lutonix; BD Bard, Covington, Ga) after atherectomy. Mean lesion length was 13.2 ± 9.0 cm. Thirty-one (58%) lesions were TransAtlantic Inter-Society Consensus C or D class. At 1-month follow-up, 39 of 45 (87%) patients experienced improvement in symptoms and Rutherford class. An improvement in ankle-brachial index was also noted in 13% of patients without improvement of symptoms, with no patients progressing to surgical bypass or major amputation. Mean ankle-brachial index increased from 0.54 ± 0.035 to 0.90 ± 0.031 at 1 month after intervention (P < .001) and remained constant out to 18 months. Mean toe pressure increased from 36 ± 3.8 mm Hg to 67 ± 4.5 mm Hg at 1 month after intervention (P < .001) and remained constant out to 18 months. Kaplan-Meier primary patency rate was 75% (95% confidence interval, 61%-85%) at 12 months and 65% (51%-77%) at 24 months. There was a trend toward improved primary patency after adjunctive DCBA compared with plain balloon angioplasty at 1 year (75% vs 43%; P = .1082). There was no significant difference in mortality between adjunctive DCBA and plain balloon angioplasty at 2 years (11% vs 0%). The 2-year incidence of major amputation in critical limb-threatening ischemia patients was 3.9% (1.2%-6.5%). One patient died and none underwent amputation within 30 days. CONCLUSIONS: Rotational atherectomy with adjunctive DCBA of long SFA lesions has excellent long-term patency. Two-year major amputation and mortality rates are low, and the technique has an exceptional safety profile.

Geographical Differences in Cardiovascular Comorbidities and Outcomes of COVID-19 Hospitalized Patients in the USA.

INTRODUCTION: Cardiovascular comorbidities may predispose to adverse outcomes in hospitalized patients with coronavirus disease 2019 (COVID-19). However, across the USA, the burden of cardiovascular comorbidities varies significantly. Whether clinical outcomes of hospitalized patients with COVID-19 differ between regions has not yet been studied systematically. Here, we report differences in underlying cardiovascular comorbidities and clinical outcomes of patients hospitalized with COVID-19 in Texas and in New York state. METHODS: We established a multicenter retrospective registry including patients hospitalized with COVID-19 between March 15 and July 12, 2020. Demographic and clinical data were manually retrieved from electronic medical records. We focused on the following outcomes: mortality, need for pharmacologic circulatory support, need for mechanical ventilation, and need for hemodialysis. Univariate and multivariate logistic regression analyses were performed. RESULTS: Patients in the Texas cohort (n = 296) were younger (57 vs. 63 years, p value <0.001), they had a higher BMI (30.3 kg/m2 vs. 28.5 kg/m2, p = 0.015), and they had higher rates of diabetes mellitus (41 vs. 30%; p = 0.014). In contrast, patients in the New York state cohort (n = 218) had higher rates of coronary artery disease (19 vs. 10%, p = 0.005) and atrial fibrillation (11 vs. 5%, p = 0.012). Pharmacologic circulatory support, mechanical ventilation, and hemodialysis were more frequent in the Texas cohort (21 vs. 13%, p = 0.020; 30 vs. 12%, p < 0.001; and 11 vs. 5%, p = 0.009, respectively). In-hospital mortality was similar between the 2 cohorts (16 vs. 18%, p = 0.469). After adjusting for differences in underlying comorbidities, only the use of mechanical ventilation remained significantly higher in the participating Texas hospitals (odds ratios [95% CI]: 3.88 [1.23, 12.24]). Median time to pharmacologic circulatory support was 8 days (interquartile range: 2, 13.8) in the Texas cohort compared to 1 day (0, 3) in the New York state cohort, while median time to in-hospital mortality was 16 days (10, 25.5) and 7 days (4, 14), respectively (both p < 0.001). In-hospital mortality was higher in the late versus the early study phase in the New York state cohort (24 vs. 14%, p = 0.050), while it was similar between the 2 phases in the Texas cohort (16 vs. 15%, p = 0.741). CONCLUSIONS: Geographical differences, including practice pattern variations and the impact of disease burden on provision of health care, are important for the evaluation of COVID-19 outcomes. Unadjusted data may cause bias affecting future regulatory policies and proper allocation of resources.

PrimPol, an archaic primase/polymerase operating in human cells.

We describe a second primase in human cells, PrimPol, which has the ability to start DNA chains with deoxynucleotides unlike regular primases, which use exclusively ribonucleotides. Moreover, PrimPol is also a DNA polymerase tailored to bypass the most common oxidative lesions in DNA, such as abasic sites and 8-oxoguanine. Subcellular fractionation and immunodetection studies indicated that PrimPol is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from PRIMPOL knockout mice. PRIMPOL gene silencing or ablation in human and mouse cells impaired mitochondrial DNA replication. On the basis of the synergy observed with replicative DNA polymerases Polγ and Polε, PrimPol is proposed to facilitate replication fork progression by acting as a translesion DNA polymerase or as a specific DNA primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear DNA replication.

METTL15 introduces N4-methylcytidine into human mitochondrial 12S rRNA and is required for mitoribosome biogenesis.

Post-transcriptional RNA modifications, the epitranscriptome, play important roles in modulating the functions of RNA species. Modifications of rRNA are key for ribosome production and function. Identification and characterization of enzymes involved in epitranscriptome shaping is instrumental for the elucidation of the functional roles of specific RNA modifications. Ten modified sites have been thus far identified in the mammalian mitochondrial rRNA. Enzymes responsible for two of these modifications have not been characterized. Here, we identify METTL15, show that it is the main N4-methylcytidine (m4C) methyltransferase in human cells and demonstrate that it is responsible for the methylation of position C839 in mitochondrial 12S rRNA. We show that the lack of METTL15 results in a reduction of the mitochondrial de novo protein synthesis and decreased steady-state levels of protein components of the oxidative phosphorylation system. Without functional METTL15, the assembly of the mitochondrial ribosome is decreased, with the late assembly components being unable to be incorporated efficiently into the small subunit. We speculate that m4C839 is involved in the stabilization of 12S rRNA folding, therefore facilitating the assembly of the mitochondrial small ribosomal subunits. Taken together our data show that METTL15 is a novel protein necessary for efficient translation in human mitochondria.

NSUN2 introduces 5-methylcytosines in mammalian mitochondrial tRNAs.

Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation-knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells-we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.

Effect of solid feed level and types of roughage on passage kinetics of milk replacer, concentrate, and roughage in veal calves.

This study aimed (1) to provide estimates of total mean retention times of milk replacer (MR), concentrates, and roughage in veal calves fed a mixed diet; (2) to determine the effect of level and type of solid feed (SF) on passage kinetics of MR, concentrates, and roughages in veal calves; and (3) to compare passage kinetics in veal calves using the fecal excretion curves of indigestible markers and a noninvasive 13C tracer breath test approach to determine whether the latter technique can serve as an alternative. At the start of the trial, 48 Holstein-Friesian calves (6 wk of age; 68 ± 7.7 kg of body weight; BW) were assigned to 1 of 4 dietary treatments (for statistical analysis, only 39 calf observations were used). Three treatments contained chopped wheat straw as roughage in the SF mixture in a concentrate:roughage ratio of 90:10 (dry matter basis). The SF level was 20 g/kg of metabolic BW per day (low straw), 30 g/kg of metabolic BW per day (middle straw), or 40 g/kg of metabolic BW per day (high straw). The fourth treatment (high hay) contained long perennial ryegrass hay as roughage in the SF mixture in a concentrate:roughage ratio of 70:30 (dry matter basis, at 40 g/kg of metabolic BW per day). The quantity of MR was fixed for the high straw treatment, whereas the amount of MR for the other treatments during the adaptation period was adjusted based on a pair gain strategy (i.e., exchanging ration components but keeping similar net energy). At the end of the adaptation period, calves ranged from 12 to 15 wk of age with an average BW of 123 ± 8.6 kg. Passage kinetics of concentrates were estimated by measuring 13C enrichment excess of CO2 in breath from a pulsed-dose of [1-13C]octanoate. Passage kinetics of roughage, concentrates, and MR were also estimated using fecal excretion curves obtained after ingestion of chromium-mordanted roughage, Yb2O3, and Co-EDTA, respectively. We conclude that [1-13C]octanoate cannot serve as a measure for oro-duodenal transit of concentrates because of unrealistic estimates. Based on the fecal excretion curves, we concluded that the total mean retention time of MR (i.e., time to peak; the moment that the excretion curve reaches peak concentration) was, on average, 12.4 h, and that the passage kinetics of MR was not affected by the level or type of SF. The mean retention time of concentrates was shorter (21.4 h) than that of both straw (59.1 h) and hay (36.8 h), and was not affected by the level or type of SF. Also, the mean retention time of the slowest compartment (i.e., the rumen) was shorter for concentrates (39.6 h) than that of straw (110.0 h) and hay (59.2 h). Contrary, the passage of roughage was affected by level and type of SF. Long hay increased time to peak by 22.3 h and decreased ruminal mean retention time by 50.8 h relative to chopped straw, indicating that the passage rate of long hay is faster than that of chopped straw. We conclude that the level and type of SF only affects the passage kinetics of roughage and not that of MR and concentrates.

Reconstruction of the Proximal Scaphoid With a Medial Femoral Trochlea Osteochondral Graft: Minimum 2-Year Results.

PURPOSE: To report the clinical and radiological outcomes after medial femoral trochlear (MFT) osteochondral graft for the salvage of proximal scaphoid fractures with a minimum 2-year follow-up. METHODS: A retrospective review was performed of patients with comminuted fractures of the proximal scaphoid treated by excision of the proximal pole and replacement with free vascularized MFT osteochondral graft. Demographic data, objective and radiographic measurements, and patient-reported outcome measures of the upper limb and knee were collected. Pain was assessed by completion of a visual analog scale (VAS). RESULTS: Between February 2014 and May 2015, 12 MFT osteochondral grafts were performed. Eight patients were available for follow-up at a mean of 34 months (range, 28-39 months). The mean range of wrist flexion was 31° (range, 15°-60°), extension was 34° (range, 5°-60°), radial deviation was 9° (range, 0°-20°), ulnar deviation was 28° (range, 10°-45°) and grip strength was 42 kg (range, 25-53 kg). The median wrist pain, as measured by VAS, was 0.7 (mean, 1.3; range, 0-6). The average follow-up scapholunate, radiolunate, and radioscaphoid angles were 58.9° (range, 44°-93°), 12.9° (range, 0°-30°), and 46.0° (range, 35°-63°), respectively. The mean Disabilities of the Arm, Shoulder, and Hand (DASH) score was 13.9 (range, 3-43) and Patient Rated Wrist Evaluation (PRWE) score was 22.4 (range, 2-68). The mean postoperative Oxford Knee Score was 42 (range, 14-48). One patient suffered notable knee pain at 37-month follow-up. One patient suffered notable pain on the radial side of the wrist and underwent scaphoid excision and 4-corner arthrodesis. CONCLUSIONS: Replacement of the fragmented proximal scaphoid by MFT graft is an alternative to other salvage options and most patients can expect pain relief and acceptable wrist motion. These results need to be balanced against the potential for donor-site morbidity. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic V.

TRMT2B is responsible for both tRNA and rRNA m5U-methylation in human mitochondria.

RNA species play host to a plethora of post-transcriptional modifications which together make up the epitranscriptome. 5-methyluridine (m5U) is one of the most common modifications made to cellular RNA, where it is found almost ubiquitously in bacterial and eukaryotic cytosolic tRNAs at position 54. Here, we demonstrate that m5U54 in human mitochondrial tRNAs is catalysed by the nuclear-encoded enzyme TRMT2B, and that its repertoire of substrates is expanded to ribosomal RNAs, catalysing m5U429 in 12S rRNA. We show that TRMT2B is not essential for viability in human cells and that knocking-out the gene shows no obvious phenotype with regards to RNA stability, mitochondrial translation, or cellular growth.

Patients with chronic mild or moderate traumatic brain injury have abnormal brain enlargement.

Introduction: Much less is known about brain volume abnormalities in patients with chronic mild or moderate traumatic brain injury (TBI) compared with patients with more severe injury. Commercially available software methods including NeuroQuant® are being used increasingly to assess MRI brain volume in patients with TBI.Methods: 50 patients with mild or moderate TBI were compared to the NeuroQuant® normal control database (n = thousands) with respect to MRI brain volume.Results: The patients had many areas of abnormal enlargement and fewer areas of atrophy, including abnormally small cerebral white matter (CWM) limited to the first 10 months after injury. Examination of correlations within the patient group between CWM volume and volumes of the abnormally enlarged regions showed multiple significant negative correlations, indicating that CWM atrophy correlated with enlargement of the other regions.Discussion: The finding of many regions of abnormal brain enlargement was relatively new, although a couple of previous studies of patients with mild TBI found similar but more limited findings. The cause of the abnormal enlargement was unknown, but possibilities included: (1) hyperactivity and hypertrophy; or (2) chronic neuro-inflammation and edema.Abbreviations: ADNI: Alzheimer's Disease Neuroimaging Initiative; CWM: cerebral white matter; GM: cerebral cortical gray matter; ICC: intraclass correlations coefficient; IFT: infratentorial; MRI: magnetic resonance imaging; mTBI: mild TBI; NQ: NeuroQuant®; SCN: subcortical nuclei; t0: time of injury; t1: time of first NeuroQuanted MRI scan after injury; t2: time of second NeuroQuanted MRI scan after injury; TBI: traumatic brain injury; VBR: ventricle-to-brain ratio; WBP: whole-brain parenchyma.

Mutations in ELAC2 associated with hypertrophic cardiomyopathy impair mitochondrial tRNA 3'-end processing.

Mutations in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, responsible for endonucleolytic cleavage of the 3' ends of mitochondrial pre-tRNAs. Here, we report the identification of 16 novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy (HCM), and lactic acidosis. We provide evidence for the pathogenicity of the novel missense variants by studying the RNase Z activity in an in vitro system. We also modeled the residues affected by a missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the affected individuals have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile-onset forms of HCM and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, suggesting a functional link between tumorigenesis and mitochondrial RNA metabolism.

Elementary functions modified for seasonal effects to describe growth in freshwater fish✰.

Two models were derived to describe fish growth while accounting for the effects of fluctuating water temperatures. The models were initially expressed in a rate:state form and subsequently integrated resulting in two analytical solutions, representing two distinct types of growth: exponential (Model 1) and asymptotic (Model 2). Both models share the assumptions that growth machinery works at a rate which varies with water temperature and that growth is irreversible. In addition, in Model 1 it is assumed that quantity of growth machinery is proportional to live body weight and substrate is non-limiting over the period of growth; whereas Model 2 is based on the assumption that quantity of growth machinery is proportional to available substrate. Effects of seasonal variations in water temperature on fish growth are represented in both models by a sinusoidal function. The potential of these models was investigated through their ability to describe growth in eight datasets encompassing three species: European bullhead (Cottus gobio), brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss). Models were evaluated using statistical measures of goodness-of-fit and through the analysis of residuals. Of the eight datasets, six displayed asymptotic growth while the other two exhibited exponential growth. Both models yield suitable simple growth functions with acceptable goodness-of-fit to fish growth curves under fluctuating water temperatures. However, Model 1, representing exponential growth, shows limited ability to predict fish size (length) when growth curves follow a clear asymptotic trend. This study enforces the idea that a given model is not always superior to another and that data structure and underlying model assumptions must be considered in model selection.

Human mitochondrial ribosomes can switch their structural RNA composition.

The recent developments in cryo-EM have revolutionized our access to previously refractory structures. In particular, such studies of mammalian mitoribosomes have confirmed the absence of any 5S rRNA species and revealed the unexpected presence of a mitochondrially encoded tRNA (mt-tRNA) that usurps this position. Although the cryo-EM structures resolved the conundrum of whether mammalian mitoribosomes contain a 5S rRNA, they introduced a new dilemma: Why do human and porcine mitoribosomes integrate contrasting mt-tRNAs? Human mitoribosomes have been shown to integrate mt-tRNAVal compared with the porcine use of mt-tRNAPhe We have explored this observation further. Our studies examine whether a range of mt-tRNAs are used by different mammals, or whether the mt-tRNA selection is strictly limited to only these two species of the 22 tRNAs encoded by the mitochondrial genome (mtDNA); whether there is tissue-specific variation within a single organism; and what happens to the human mitoribosome when levels of the mt-tRNAVal are depleted. Our data demonstrate that only mt-tRNAVal or mt-tRNAPhe are found in the mitoribosomes of five different mammals, each mammal favors the same mt-tRNA in all tissue types, and strikingly, when steady-state levels of mt-tRNAVal are reduced, human mitoribosome biogenesis displays an adaptive response by switching to the incorporation of mt-tRNAPhe to generate translationally competent machinery.

The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules.

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNAMet, and mt-tRNAPhe, and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression.

TRMT5 Mutations Cause a Defect in Post-transcriptional Modification of Mitochondrial tRNA Associated with Multiple Respiratory-Chain Deficiencies.

Deficiencies in respiratory-chain complexes lead to a variety of clinical phenotypes resulting from inadequate energy production by the mitochondrial oxidative phosphorylation system. Defective expression of mtDNA-encoded genes, caused by mutations in either the mitochondrial or nuclear genome, represents a rapidly growing group of human disorders. By whole-exome sequencing, we identified two unrelated individuals carrying compound heterozygous variants in TRMT5 (tRNA methyltransferase 5). TRMT5 encodes a mitochondrial protein with strong homology to members of the class I-like methyltransferase superfamily. Both affected individuals presented with lactic acidosis and evidence of multiple mitochondrial respiratory-chain-complex deficiencies in skeletal muscle, although the clinical presentation of the two affected subjects was remarkably different; one presented in childhood with failure to thrive and hypertrophic cardiomyopathy, and the other was an adult with a life-long history of exercise intolerance. Mutations in TRMT5 were associated with the hypomodification of a guanosine residue at position 37 (G37) of mitochondrial tRNA; this hypomodification was particularly prominent in skeletal muscle. Deficiency of the G37 modification was also detected in human cells subjected to TRMT5 RNAi. The pathogenicity of the detected variants was further confirmed in a heterologous yeast model and by the rescue of the molecular phenotype after re-expression of wild-type TRMT5 cDNA in cells derived from the affected individuals. Our study highlights the importance of post-transcriptional modification of mitochondrial tRNAs for faithful mitochondrial function.