Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish

Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. The import of some, but not all, nuclear-encoded mitochondrial proteins begins with translation of messenger RNAs (mRNAs) on the surface of mitochondria. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a-/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to employ localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.

Molecular Changes in Prader-Willi Syndrome Neurons Reveals Clues About Increased Autism Susceptibility

Background: Prader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by hormonal dysregulation, obesity, intellectual disability, and behavioral problems. Most PWS cases are caused by paternal interstitial deletions of 15q11.2-q13.1, while a smaller number of cases are caused by chromosome 15 maternal uniparental disomy (PW-UPD). Children with PW-UPD are at higher risk for developing autism spectrum disorder (ASD) than the neurotypical population. In this study, we used expression analysis of PW-UPD neurons to try to identify the molecular cause for increased autism risk.Methods: Dental pulp stem cells (DPSC) from neurotypical control and PWS subjects were differentiated to neurons for mRNA sequencing. Significantly differentially expressed transcripts among all groups were identified. Downstream protein analysis including immunocytochemistry and immunoblots were performed to confirm the transcript level data and pathway enrichment findings.Results: We identified 9 transcripts outside of the PWS critical region (15q11.2-q13.1) that may contribute to core PWS phenotypes. Moreover, we discovered a global reduction in mitochondrial transcripts in the PW-UPD + ASD group. We also found decreased mitochondrial abundance along with mitochondrial aggregates in the cell body and neural projections of +ASD neurons.Conclusion: The 9 transcripts we identified common to all PWS subtypes may reveal PWS specific defects during neurodevelopment. Importantly, we found a global reduction in mitochondrial transcripts in PW-UPD + ASD neurons versus control and other PWS subtypes. We then confirmed mitochondrial defects in neurons from individuals with PWS at the cellular level. Quantification of this phenotype supports our hypothesis that the increased incidence of ASD in PW-UPD subjects may arise from mitochondrial defects in developing neurons.

Structural basis of RNA processing by human mitochondrial RNase P

Human mitochondrial transcripts contain messenger and ribosomal RNAs flanked by transfer RNAs (tRNAs), which are excised by mitochondrial RNase (mtRNase) P and Z to liberate all RNA species. In contrast to nuclear or bacterial RNase P, mtRNase P is not a ribozyme but comprises three protein subunits that carry out RNA cleavage and methylation by unknown mechanisms. Here, we present the cryo-EM structure of human mtRNase P bound to precursor tRNA, which reveals a unique mechanism of substrate recognition and processing. Subunits TRMT10C and SDR5C1 form a subcomplex that binds conserved mitochondrial tRNA elements, including the anticodon loop, and positions the tRNA for methylation. The endonuclease PRORP is recruited and activated through interactions with its PPR and nuclease domains to ensure precise pre-tRNA cleavage. The structure provides the molecular basis for the first step of RNA processing in human mitochondria.

Quality control in scRNA-Seq can discriminate pacemaker cells: the mtRNA bias

Single-cell RNA-sequencing (scRNA-seq) provides high-resolution insights into complex tissues. Cardiac tissue, however, poses a major challenge due to the delicate isolation process and the large size of mature cardiomyocytes. Regardless of the experimental technique, captured cells are often impaired and some capture sites may contain multiple or no cells at all. All this refers to “low quality” potentially leading to data misinterpretation. Common standard quality control parameters involve the number of detected genes, transcripts per cell, and the fraction of transcripts from mitochondrial genes. While cutoffs for transcripts and genes per cell are usually user-defined for each experiment or individually calculated, a fixed threshold of 5% mitochondrial transcripts is standard and often set as default in scRNA-seq software. However, this parameter is highly dependent on the tissue type. In the heart, mitochondrial transcripts comprise almost 30% of total mRNA due to high energy demands. Here, we demonstrate that a 5%-threshold not only causes an unacceptable exclusion of cardiomyocytes but also introduces a bias that particularly discriminates pacemaker cells. This effect is apparent for our in vitro generated induced-sinoatrial-bodies (iSABs; highly enriched physiologically functional pacemaker cells), and also evident in a public data set of cells isolated from embryonal murine sinoatrial node tissue (Goodyer William et al. in Circ Res 125:379–397, 2019). Taken together, we recommend omitting this filtering parameter for scRNA-seq in cardiovascular applications whenever possible.

Molecular Changes in Prader-Willi Syndrome Neurons Reveals Clues About Increased Autism Susceptibility.

Background: Prader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by hormonal dysregulation, obesity, intellectual disability, and behavioral problems. Most PWS cases are caused by paternal interstitial deletions of 15q11.2-q13.1, while a smaller number of cases are caused by chromosome 15 maternal uniparental disomy (PW-UPD). Children with PW-UPD are at higher risk for developing autism spectrum disorder (ASD) than the neurotypical population. In this study, we used expression analysis of PW-UPD neurons to try to identify the molecular cause for increased autism risk. Methods: Dental pulp stem cells (DPSC) from neurotypical control and PWS subjects were differentiated to neurons for mRNA sequencing. Significantly differentially expressed transcripts among all groups were identified. Downstream protein analysis including immunocytochemistry and immunoblots were performed to confirm the transcript level data and pathway enrichment findings. Results: We identified 9 transcripts outside of the PWS critical region (15q11.2-q13.1) that may contribute to core PWS phenotypes. Moreover, we discovered a global reduction in mitochondrial transcripts in the PW-UPD +ASD group. We also found decreased mitochondrial abundance along with mitochondrial aggregates in the cell body and neural projections of +ASD neurons. Conclusions: The 9 transcripts we identified common to all PWS subtypes may reveal PWS specific defects during neurodevelopment. Importantly, we found a global reduction in mitochondrial transcripts in PW-UPD +ASD neurons versus control and other PWS subtypes. We then confirmed mitochondrial defects in neurons from individuals with PWS at the cellular level. Quantification of this phenotype supports our hypothesis that the increased incidence of ASD in PW-UPD subjects may arise from mitochondrial defects in developing neurons.

PPR-DYW Protein EMP17 Is Required for Mitochondrial RNA Editing, Complex III Biogenesis, and Seed Development in Maize

The conversion of cytidines to uridines (C-to-U) at specific sites in mitochondrial and plastid transcripts is a post-transcriptional processing event that is important to the expression of organellar genes. Pentatricopeptide repeat (PPR) proteins are involved in this process. In this study, we report the function of a previously uncharacterized PPR-DYW protein, Empty pericarp17 (EMP17), in the C-to-U editing and kernel development in maize. EMP17 is targeted to mitochondria. The loss-function of EMP17 arrests maize kernel development, abolishes the editing at ccmFC-799 and nad2-677 sites, and reduces the editing at ccmFC-906 and -966 sites. The absence of editing causes amino acid residue changes in CcmFC-267 (Ser to Pro) and Nad2-226 (Phe to Ser), respectively. As CcmFC functions in cytochrome c (Cytc) maturation, the amount of Cytc and Cytc1 protein is drastically reduced in emp17, suggesting that the CcmFC-267 (Ser to Pro) change impairs the CcmFC function. As a result, the assembly of complex III is strikingly decreased in emp17. In contrast, the assembly of complex I appears less affected, suggesting that the Nad2-226 (Phe to Ser) change may have less impact on Nad2 function. Together, these results indicate that EMP17 is required for the C-to-U editing at several sites in mitochondrial transcripts, complex III biogenesis, and seed development in maize.

Unravelling the impact of intrauterine growth restriction on heart development: insights into mitochondria and sexual dimorphism from a non-hominoid primate

Fetal exposure to an unfavorable intrauterine environment programs an individual to have a greater susceptibility later in life to non-communicable diseases, such as coronary heart disease, but the molecular processes are poorly understood. An article in Clinical Science recently reported novel details on the effects of maternal nutrient reduction (MNR) on fetal heart development using a primate model that is about 94% genetically similar to humans and is also mostly monotocous. MNR adversely impacted fetal left ventricular (LV) mitochondria in a sex-dependent fashion with a greater effect on male fetuses, although mitochondrial transcripts increased more so in females. Increased expression for several respiratory chain and adenosine triphosphate (ATP) synthase proteins were observed. However, fetal LV mitochondrial complex I and complex II/III activities were significantly decreased, likely contributing to a 73% decreased LV ATP content and increased LV lipid peroxidation. Moreover, MNR fetal LV mitochondria showed sparse and disarranged cristae. This study indicates that mitochondria are targets of the remodeling and imprinting processes in a sex-dependent manner. Mitochondrial ROS production and inadequate energy production add another layer of complexity. Altogether these observations raise the possibility that dysfunctional mitochondria in the fetus may contribute in turn to epigenetic memory of in utero stress in the adult. The role of mitoepigenetics and involvement of mitochondrial and genomic non-coding RNAs in mitochondrial functions and nuclei–mitochondria crosstalk with in utero stress awaits further investigation.

Ribosome Profiling Reveals a Dichotomy Between Ribosome Occupancy of Nuclear-Encoded and Mitochondrial-Encoded OXPHOS mRNA Transcripts in a Striatal Cell Model of Huntington Disease

Huntington disease (HD) is caused by an expanded polyglutamine mutation in huntingtin (mHTT), which promotes a prominent atrophy in the striatum and subsequent psychiatric, cognitive, and choreiform movements. Multiple lines of evidence point to an association between HD and aberrant striatal mitochondrial functions. However, present knowledge about whether (or how) mitochondrial mRNA translation is differentially regulated in HD remains unclear. We have recently applied ribosome profiling (Ribo-Seq), a technique based on the high-throughput sequencing of ribosome-protected mRNA fragments, to analyze detailed snapshots of ribosome occupancy of the mitochondrial mRNA transcripts in control and HD striatal cells. Ribo-seq data revealed almost unaltered ribosome occupancy on the nuclear-encoded mitochondrial transcripts involved in oxidative phosphorylation (OXPHOS) and only a mild reduction in ribosome occupancy on a few selected transcripts (SHDA, Ndufv1, Timm23, Tomm5, and Mrps22) in HD cells. By contrast, ribosome occupancy of mitochondrially encoded OXPHOS mRNAs (mtNd-1, mtNd-2, mtNd-4, mtNd-4l, mtNd-5, mtNd-6, mt-Co1, mtCyt b, and mt-ATP8) was dramatically increased, implying widespread dichotomous effects on ribosome occupancy and OXPHOS mRNA translation in HD. Thus, mHTT may command signals that specifically regulate translation of the mitochondrial OXPHOS transcripts and influence HD pathogenesis.

Heat stress reduces maturation and developmental capacity in bovine oocytes

The ovarian pool of follicles, and their enclosed oocytes, is highly sensitive to hyperthermia. Heat-induced changes in small antral follicles can later manifest as impaired follicle development and compromised competence of the enclosed oocytes to undergo maturation, fertilisation and further development into an embryo. This review describes the main changes documented so far that underlie the oocyte damage. The review discusses some cellular and molecular mechanisms by which heat stress compromises oocyte developmental competence, such as impairment of nuclear and cytoplasmic maturation and mitochondrial function, changes in the expression of both nuclear and mitochondrial transcripts and the induction of apoptosis. The review emphasises that although the oocyte is exposed to heat stress, changes are also evident in the developed embryo. Moreover, the effect of heat stress is not limited to the summer; it carries over to the cold autumn, as manifest by impaired steroid production, low oocyte competence and reduced fertility. The spontaneous recovery of oocytes from the end of the summer through the autumn until the beginning of winter suggests that only subpopulations of follicles, rather than the entire ovarian reserve, are damaged upon heat exposure.

Differential RNA Editing and Intron Splicing in Soybean Mitochondria during Nodulation

Nitrogen fixation in soybean consumes a tremendous amount of energy, leading to substantial differences in energy metabolism and mitochondrial activities between nodules and uninoculated roots. While C-to-U RNA editing and intron splicing of mitochondrial transcripts are common in plant species, their roles in relation to nodule functions are still elusive. In this study, we performed RNA-seq to compare transcript profiles and RNA editing of mitochondrial genes in soybean nodules and roots. A total of 631 RNA editing sites were identified on mitochondrial transcripts, with 12% or 74 sites differentially edited among the transcripts isolated from nodules, stripped roots, and uninoculated roots. Eight out of these 74 differentially edited sites are located on the matR transcript, of which the degrees of RNA editing were the highest in the nodule sample. The degree of mitochondrial intron splicing was also examined. The splicing efficiencies of several introns in nodules and stripped roots were higher than in uninoculated roots. These include nad1 introns 2/3/4, nad4 intron 3, nad5 introns 2/3, cox2 intron 1, and ccmFc intron 1. A greater splicing efficiency of nad4 intron 1, a higher NAD4 protein abundance, and a reduction in supercomplex I + III2 were also observed in nodules, although the causal relationship between these observations requires further investigation.