Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play important roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focus on exploring the function of GTPBP8, the human homolog of EngB. We find that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells shows the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis reveals that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit 16 S rRNA. Our study highlights the role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation.

Mitochondrial dysfunction can elicit multiple inflammatory pathways, especially when apoptotic caspases are inhibited. Such an inflammatory program is negatively regulated by the autophagic disposal of permeabilized mitochondria. Recent data demonstrate that the ubiquitination of mitochondrial proteins is essential for NEMO-driven NF-kB activation downstream of mitochondrial permeabilization.

Small peptides have previously been reported to be encoded in mitochondrial rRNA and translated by cytosolic ribosomes. In this issue of Cell Metabolism, Hu et al. use mass spectrometry to identify a cytosolically translated protein, encoded instead in mitochondrial mRNA, that is surprisingly targeted back into the mitochondrial matrix.

We investigated the impact of a rumen-bypass protein (RBP) supplement on growth performance, plasma and urinary N (UN) concentration, hepatic mitochondrial protein complexes, and hepatic mRNA expression of immune genes of beef steers with negative or positive residual feed intake (RFI) phenotype. Forty crossbred beef steers with an average body weight (BW) of 492 ± 36 kg were subjected to a generalized randomized block design over a 42-day experimental period. This study followed a 2 × 2 factorial arrangement of treatments. The factors evaluated were: 1) RFI classification (low-RFI (-2.12 kg/d) vs. high-RFI (2.02 kg/d), and 2) rumen-bypass protein supplement: RBP supplement (RBP; 227 g/steer/d) vs. control diet (CON; 0 g/d), resulting in four distinct treatments: LRFI-CON (n = 10), LRFI-RBP (n = 10), HRFI-CON (n = 10), and HRFI-RBP (n = 10). The RBP supplement (84% crude protein) is a mixture of hydrolyzed feather meal, porcine blood meal, and DL-methionine hydroxy analogue. The beef steers were stratified by BW, randomly assigned to treatments, and housed in four pens (1 treatment/pen) equipped with two GrowSafe feed bunks each to measure individual dry mater intake (DMI). Body weight was measured every 7 d. Liver tissue samples were collected on d 42 from all the beef steers. These samples were used for mRNA expression analysis of 16 immune-related genes and for evaluating the mitochondrial protein complexes I - V. No significant effects due to RBP supplementation or RFI × RBP interactions (P > 0.05) were observed for average daily gain (ADG) and DMI. However, compared to high-RFI steers, low-RFI steers showed a trend towards reduced DMI (12.9 vs. 13.6 kg/d; P = 0.07) but ADG was similar for the two RFI groups. Regardless of RFI status, supplemental RBP increased blood urea nitrogen (BUN) (P = 0.01), with a lower BUN concentration in low-RFI steers compared to high-RFI ones. A tendency for interaction (P = 0.07) between RFI and RBP was detected for the UN concentrations; feeding the dietary RBP increased the UN concentration in high-RFI beef steers (209 vs. 124 mM), whereas the concentration was lower than that of the CON group for low-RFI beef steers (86 vs. 131 mM). Interactions of RBP and RFI were observed (P ≤ 0.05) for mitochondrial activities of complexes IV, V, and mRNA expressions of some immune genes such as TLR2, TLR3, and IL23A. In conclusion, while RBP supplementation did not alter growth performance, its observed effects on hepatic immune gene expression, mitochondrial protein complexes, BUN, and UN depended on the beef steers\' RFI phenotype. Therefore, the RFI status of beef steers should be considered in future studies evaluating the effects of dietary protein supplements.

In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner.

Mitochondrial shape and network formation have been primarily associated with the well-established processes of fission and fusion. However, recent research has unveiled an intricate and multifaceted landscape of mitochondrial morphology that extends far beyond the conventional fission-fusion paradigm. These less-explored dimensions harbor numerous unresolved mysteries. This review navigates through diverse processes influencing mitochondrial shape and network formation, highlighting the intriguing complexities and gaps in our understanding of mitochondrial architecture. The exploration encompasses various scales, from biophysical principles governing membrane dynamics to molecular machineries shaping mitochondria, presenting a roadmap for future research in this evolving field.

Mitochondria-related neurodegenerative diseases have been implicated in the disruption of primary cilia function. Mutation in an intrinsic mitochondrial complex I component NDUFAF2 has been identified in Leigh syndrome, a severe inherited mitochondriopathy. Mutations in ARMC9, which encodes a basal body protein, cause Joubert syndrome, a ciliopathy with defects in the brain, kidney, and eye. Here, we report a mechanistic link between mitochondria metabolism and primary cilia signaling. We discovered that loss of NDUFAF2 caused both mitochondrial and ciliary defects in vitro and in vivo and identified NDUFAF2 as a binding partner for ARMC9. We also found that NDUFAF2 was both necessary and sufficient for cilia formation and that exogenous expression of NDUFAF2 rescued the ciliary and mitochondrial defects observed in cells from patients with known ARMC9 deficiency. NAD+ supplementation restored mitochondrial and ciliary dysfunction in ARMC9-deficient cells and zebrafish and ameliorated the ocular motility and motor deficits of a patient with ARMC9 deficiency. The present results provide a compelling mechanistic link, supported by evidence from human studies, between primary cilia and mitochondrial signaling. Importantly, our findings have significant implications for the development of therapeutic approaches targeting ciliopathies.

Rationale: Autophagy dysregulation is known to be a mechanism of doxorubicin (DOX)-induced cardiotoxicity (DIC). Mitochondrial-Endoplasmic Reticulum Contacts (MERCs) are where autophagy initiates and autophagosomes form. However, the role of MERCs in autophagy dysregulation in DIC remains elusive. FUNDC1 is a tethering protein of MERCs. We aim to investigate the effect of DOX on MERCs in cardiomyocytes and explore whether it is involved in the dysregulated autophagy in DIC. Methods: We employed confocal microscopy and transmission electron microscopy to assess MERCs structure. Autophagic flux was analyzed using the mCherry-EGFP-LC3B fluorescence assay and western blotting for LC3BII. Mitophagy was studied through the mCherry-EGFP-FIS1 fluorescence assay and colocalization analysis between LC3B and mitochondria. A total dose of 18 mg/kg of doxorubicin was administrated in mice to construct a DIC model in vivo. Additionally, we used adeno-associated virus (AAV) to cardiac-specifically overexpress FUNDC1. Cardiac function and remodeling were evaluated by echocardiography and Masson\'s trichrome staining, respectively. Results: DOX blocked autophagic flux by inhibiting autophagosome biogenesis, which could be attributed to the downregulation of FUNDC1 and disruption of MERCs structures. FUNDC1 overexpression restored the blocked autophagosome biogenesis by maintaining MERCs structure and facilitating ATG5-ATG12/ATG16L1 complex formation without altering mitophagy. Furthermore, FUNDC1 alleviated DOX-induced oxidative stress and cardiomyocytes deaths in an autophagy-dependent manner. Notably, cardiac-specific overexpression of FUNDC1 protected DOX-treated mice against adverse cardiac remodeling and improved cardiac function. Conclusions: In summary, our study identified that FUNDC1-meditated MERCs exerted a cardioprotective effect against DIC by restoring the blocked autophagosome biogenesis. Importantly, this research reveals a novel role of FUNDC1 in enhancing macroautophagy via restoring MERCs structure and autophagosome biogenesis in the DIC model, beyond its previously known regulatory role as an mitophagy receptor.

Mitochondrial transcription factor A (TFAM) employs DNA bending to package mitochondrial DNA (mtDNA) into nucleoids and recruit mitochondrial RNA polymerase (POLRMT) at specific promoter sites, light strand promoter (LSP) and heavy strand promoter (HSP). Herein, we characterize the conformational dynamics of TFAM on promoter and non-promoter sequences using single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule protein-induced fluorescence enhancement (smPIFE) methods. The DNA-TFAM complexes dynamically transition between partially and fully bent DNA conformational states. The bending/unbending transition rates and bending stability are DNA sequence-dependent-LSP forms the most stable fully bent complex and the non-specific sequence the least, which correlates with the lifetimes and affinities of TFAM with these DNA sequences. By quantifying the dynamic nature of the DNA-TFAM complexes, our study provides insights into how TFAM acts as a multifunctional protein through the DNA bending states to achieve sequence specificity and fidelity in mitochondrial transcription while performing mtDNA packaging.

Iron deficiency is the number one nutritional problem worldwide. Iron uptake is regulated at the intestine and is highly influenced by the gut microbiome. Blood from the intestines drains directly into the liver, informing iron status and gut microbiota status. Changes in either iron or the microbiome are tightly correlated with the development of metabolic dysfunction-associated steatotic liver disease (MASLD). To investigate the underlying mechanisms of the development of MASLD that connect altered iron metabolism and gut microbiota, we compared specific pathogen free (SPF) or germ-free (GF) mice, fed a normal or low-iron diet. SPF mice on a low-iron diet showed reduced serum triglycerides and MASLD. In contrast, GF low-iron diet-fed mice showed increased serum triglycerides and did not develop hepatic steatosis. SPF mice showed significant changes in liver lipid metabolism and increased insulin resistance that was dependent upon the presence of the gut microbiota. We report that total body loss of mitochondrial iron importer Mitoferrin2 (Mfrn2-/-) exacerbated the development of MASLD on a low-iron diet with significant lipid metabolism alterations. Our study demonstrates a clear contribution of the gut microbiome, dietary iron, and Mfrn2 in the development of MASLD and metabolic syndrome.