Antigen receptor stimulation induces purifying selection against pathogenic mitochondrial tRNA mutations.

Pathogenic mutations in mitochondrial (mt) tRNA genes that compromise oxidative phosphorylation (OXPHOS) exhibit heteroplasmy and cause a range of multisyndromic conditions. Although mitochondrial disease patients are known to suffer from abnormal immune responses, how heteroplasmic mtDNA mutations affect the immune system at the molecular level is largely unknown. Here, in mice carrying pathogenic C5024T in mt-tRNAAla and in patients with mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes (MELAS) syndrome carrying A3243G in mt-tRNALeu, we found memory T and B cells to have lower pathogenic mtDNA mutation burdens than their antigen-inexperienced naive counterparts, including after vaccination. Pathogenic burden reduction was less pronounced in myeloid compared with lymphoid lineages, despite C5024T compromising macrophage OXPHOS capacity. Rapid dilution of the C5024T mutation in T and B cell cultures could be induced by antigen receptor-triggered proliferation and was accelerated by metabolic stress conditions. Furthermore, we found C5024T to dysregulate CD8+ T cell metabolic remodeling and IFN-γ production after activation. Together, our data illustrate that the generation of memory lymphocytes shapes the mtDNA landscape, wherein pathogenic variants dysregulate the immune response.

Antigen-presenting autoreactive B cells activate regulatory T cells and suppress autoimmune arthritis in mice.

B cells undergo several rounds of selection to eliminate potentially pathogenic autoreactive clones, but in contrast to T cells, evidence of positive selection of autoreactive B cells remains moot. Using unique tetramers, we traced natural autoreactive B cells (C1-B) specific for a defined triple-helical epitope on collagen type-II (COL2), constituting a sizeable fraction of the physiological B cell repertoire in mice, rats, and humans. Adoptive transfer of C1-B suppressed arthritis independently of IL10, separating them from IL10-secreting regulatory B cells. Single-cell sequencing revealed an antigen processing and presentation signature, including induced expression of CD72 and CCR7 as surface markers. C1-B presented COL2 to T cells and induced the expansion of regulatory T cells in a contact-dependent manner. CD72 blockade impeded this effect suggesting a new downstream suppressor mechanism that regulates antigen-specific T cell tolerization. Thus, our results indicate that autoreactive antigen-specific naïve B cells tolerize infiltrating T cells against self-antigens to impede the development of tissue-specific autoimmune inflammation.

Assembly of the Mitochondrial Translation Initiation Complex.

Mitochondria maintain their own translational machinery that is responsible for the synthesis of essential components of the oxidative phosphorylation system. The mammalian mitochondrial translation system differs significantly from its cytosolic and bacterial counterparts. Here, we describe detailed protocols for efficient in vitro reconstitution of the mammalian mitochondrial translation initiation complex, which can be further used for mechanistic analyses of different aspects of mitochondrial translation.

Insights into mitoribosomal biogenesis from recent structural studies.

The mitochondrial ribosome (mitoribosome) is a multicomponent machine that has unique structural features. Biogenesis of the human mitoribosome includes correct maturation and folding of the mitochondria-encoded RNA components (12S and 16S mt-rRNAs, and mt-tRNAVal) and their assembly together with 82 nucleus-encoded mitoribosomal proteins. This complex process requires the coordinated action of multiple assembly factors. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have provided detailed insights into the specific functions of several mitoribosome assembly factors and have defined their timing. In this review we summarize mitoribosomal small (mtSSU) and large subunit (mtLSU) biogenesis based on structural findings, and we discuss potential crosstalk between mtSSU and mtLSU assembly pathways as well as coordination between mitoribosome biogenesis and other processes involved in mitochondrial gene expression.

Achieving symptom relief in patients with myalgic encephalomyelitis by targeting the neuro-immune interface and optimizing disease tolerance.

Myalgic encephalomyelitis (ME) previously also known as chronic fatigue syndrome is a heterogeneous, debilitating syndrome of unknown etiology responsible for long-lasting disability in millions of patients worldwide. The most well-known symptom of ME is post-exertional malaise, but many patients also experience autonomic dysregulation, cranial nerve dysfunction and signs of immune system activation. Many patients also report a sudden onset of disease following an infection. The brainstem is a suspected focal point in ME pathogenesis and patients with structural impairment to the brainstem often show ME-like symptoms. The brainstem is also where the vagus nerve originates, a critical neuro-immune interface and mediator of the inflammatory reflex which regulate systemic inflammation. Here, we report the results of a randomized, placebo-controlled trial using intranasal mechanical stimulation targeting nerve endings in the nasal cavity, likely from the trigeminal nerve, possibly activating additional centers in the brainstem of ME patients and correlating with a ∼30% reduction in overall symptom scores after 8 weeks of treatment. By performing longitudinal, systems-level monitoring of the blood immune system in these patients, we uncover signs of chronic immune activation in ME, as well as immunological correlates of improvement that center around gut-homing immune cells and reduced inflammation. The mechanisms of symptom relief remain to be determined, but transcriptional analyses suggest an upregulation of disease tolerance mechanisms. We believe that these results are suggestive of ME as a condition explained by a maladaptive disease tolerance response following infection.

Translation initiation of leaderless and polycistronic transcripts in mammalian mitochondria.

The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It has been commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5' leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes. By using novel in vitro mitochondrial translation initiation assays, alongside biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe unique features of translation initiation in human mitochondria. We show that in vitro, leaderless mRNA transcripts can be loaded directly onto assembled 55S mitoribosomes, but not onto the mitoribosomal small subunit (28S), in a manner that requires initiator fMet-tRNAMet binding. In addition, we demonstrate that in human cells and in vitro, mtIF3 activity is not required for translation of leaderless mitochondrial transcripts but is essential for translation of ATP6 in the case of the bicistronic ATP8/ATP6 transcript. Furthermore, we show that mtIF2 is indispensable for mitochondrial protein synthesis. Our results demonstrate an important evolutionary divergence of the mitochondrial translation system and further our fundamental understanding of a process central to eukaryotic metabolism.

Human mitochondria require mtRF1 for translation termination at non-canonical stop codons.

The mitochondrial translation machinery highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG codons lacking cognate tRNAs in human mitochondria. The locations of these codons at the end of COX1 and ND6 open reading frames, respectively, suggest they might function as stop codons. However, while the canonical stop codons UAA and UAG are known to be recognized by mtRF1a, the release mechanism at AGA and AGG codons remains a debated issue. Here, we show that upon the loss of another member of the mitochondrial release factor family, mtRF1, mitoribosomes accumulate specifically at AGA and AGG codons. Stalling of mitoribosomes alters COX1 transcript and protein levels, but not ND6 synthesis. In addition, using an in vitro reconstituted mitochondrial translation system, we demonstrate the specific peptide release activity of mtRF1 at the AGA and AGG codons. Together, our results reveal the role of mtRF1 in translation termination at non-canonical stop codons in mitochondria.

Streptogramin A derivatives as mitochondrial translation inhibitors to suppress glioblastoma stem cell growth.

New therapeutic strategies for glioblastoma treatment, especially tackling the tumour's glioblastoma stem cell (GSC) component, are an urgent medical need. Recently, mitochondrial translation inhibition has been shown to affect GSC growth, clonogenicity, and self-renewal capability, therefore becoming an attractive therapeutic target. The combination of streptogramins B and A antibiotics quinupristin/dalfopristin (Q/D), which inhibits mitochondrial ribosome function, affects GSCs more effectively in vitro than the standard of care temozolomide. Here, docking calculations based on the cryo-EM structure of the Q/D-bound mitochondrial ribosome have been used to develop a series of streptogramin A derivatives. We obtained twenty-two new and known molecules starting from the dalfopristin and virginiamycin M1 scaffolds. A structure-activity relationship refinement was performed to evaluate the capability of these compounds to suppress GSC growth and inhibit mitochondrial translation, either alone or in combination with quinupristin. Finally, quantitative ultra HPLC-mass spectrometry allowed us to assess the cell penetration of some of these derivatives. Among all, the fluorine derivatives of dalfopristin and virginiamycin M1, (16R)-1e and (16R)-2e, respectively, and flopristin resulted in being more potent than the corresponding lead compounds and penetrating to a greater extent into the cells. We, therefore, propose these three compounds for further evaluation in vivo as antineoplastic agents.

Structure of the mitoribosomal small subunit with streptomycin reveals Fe-S clusters and physiological molecules.

The mitoribosome regulates cellular energy production, and its dysfunction is associated with aging. Inhibition of the mitoribosome can be caused by off-target binding of antimicrobial drugs and was shown to be coupled with a bilateral decreased visual acuity. Previously, we reported mitochondria-specific protein aspects of the mitoribosome, and in this article we present a 2.4-Å resolution structure of the small subunit in a complex with the anti-tuberculosis drug streptomycin that reveals roles of non-protein components. We found iron-sulfur clusters that are coordinated by different mitoribosomal proteins, nicotinamide adenine dinucleotide (NAD) associated with rRNA insertion, and posttranslational modifications. This is the first evidence of inter-protein coordination of iron-sulfur, and the finding of iron-sulfur clusters and NAD as fundamental building blocks of the mitoribosome directly links to mitochondrial disease and aging. We also report details of streptomycin interactions, suggesting that the mitoribosome-bound streptomycin is likely to be in hydrated gem-diol form and can be subjected to other modifications by the cellular milieu. The presented approach of adding antibiotics to cultured cells can be used to define their native structures in a bound form under more physiological conditions, and since streptomycin is a widely used drug for treatment, the newly resolved features can serve as determinants for targeting.

ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing.

Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3' phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3' phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2.

Mechanism of mitoribosomal small subunit biogenesis and preinitiation.

Mitoribosomes are essential for the synthesis and maintenance of bioenergetic proteins. Here we use cryo-electron microscopy to determine a series of the small mitoribosomal subunit (SSU) intermediates in complex with auxiliary factors, revealing a sequential assembly mechanism. The methyltransferase TFB1M binds to partially unfolded rRNA h45 that is promoted by RBFA, while the mRNA channel is blocked. This enables binding of METTL15 that promotes further rRNA maturation and a large conformational change of RBFA. The new conformation allows initiation factor mtIF3 to already occupy the subunit interface during the assembly. Finally, the mitochondria-specific ribosomal protein mS37 (ref. 1) outcompetes RBFA to complete the assembly with the SSU-mS37-mtIF3 complex2 that proceeds towards mtIF2 binding and translation initiation. Our results explain how the action of step-specific factors modulate the dynamic assembly of the SSU, and adaptation of a unique protein, mS37, links the assembly to initiation to establish the catalytic human mitoribosome.

Impaired plasma cell differentiation associates with increased oxidative metabolism in IκBNS-deficient B cells.

Mutations causing loss of the NF-κB regulator IκBNS, result in impaired development of innate-like B cells and defective plasma cell (PC) differentiation. Since productive PC differentiation requires B cell metabolic reprogramming, we sought to investigate processes important for this transition using the bumble mouse strain, deficient for IκBNS. We report that LPS-activated bumble B cells exhibited elevated mTOR activation levels, mitochondrial accumulation, increased OXPHOS and mROS production, along with a reduced capacity for autophagy, compared to wildtype B cells. Overall, our results demonstrate that PC differentiation in the absence of IκBNS is characterized by excessive activation during early rounds of B cell division, increased mitochondrial metabolism and decreased autophagic capacity, thus improving our understanding of the role of IκBNS in PC differentiation.

Structural basis for late maturation steps of the human mitoribosomal large subunit.

Mitochondrial ribosomes (mitoribosomes) synthesize a critical set of proteins essential for oxidative phosphorylation. Therefore, mitoribosomal function is vital to the cellular energy supply. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determine the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5, allowing us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis. Our structures provide essential insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16 S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangements leading to PTC formation. Moreover, our data reveal an unexpected involvement of the elongation factor mtEF-Tu in mt-LSU assembly, where mtEF-Tu interacts with GTPBP5, similar to its interaction with tRNA during translational elongation.

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.

Human Mitoribosome Biogenesis and Its Emerging Links to Disease.

Mammalian mitochondrial ribosomes (mitoribosomes) synthesize a small subset of proteins, which are essential components of the oxidative phosphorylation machinery. Therefore, their function is of fundamental importance to cellular metabolism. The assembly of mitoribosomes is a complex process that progresses through numerous maturation and protein-binding events coordinated by the actions of several assembly factors. Dysregulation of mitoribosome production is increasingly recognized as a contributor to metabolic and neurodegenerative diseases. In recent years, mutations in multiple components of the mitoribosome assembly machinery have been associated with a range of human pathologies, highlighting their importance to cell function and health. Here, we provide a review of our current understanding of mitoribosome biogenesis, highlighting the key factors involved in this process and the growing number of mutations in genes encoding mitoribosomal RNAs, proteins, and assembly factors that lead to human disease.

Inhibition of mitochondrial translation suppresses glioblastoma stem cell growth.

Glioblastoma stem cells (GSCs) resist current glioblastoma (GBM) therapies. GSCs rely highly on oxidative phosphorylation (OXPHOS), whose function requires mitochondrial translation. Here we explore the therapeutic potential of targeting mitochondrial translation and report the results of high-content screening with putative blockers of mitochondrial ribosomes. We identify the bacterial antibiotic quinupristin/dalfopristin (Q/D) as an effective suppressor of GSC growth. Q/D also decreases the clonogenicity of GSCs in vitro, consequently dysregulating the cell cycle and inducing apoptosis. Cryoelectron microscopy (cryo-EM) reveals that Q/D binds to the large mitoribosomal subunit, inhibiting mitochondrial protein synthesis and functionally dysregulating OXPHOS complexes. These data suggest that targeting mitochondrial translation could be explored to therapeutically suppress GSC growth in GBM and that Q/D could potentially be repurposed for cancer treatment.

Quantitative density gradient analysis by mass spectrometry (qDGMS) and complexome profiling analysis (ComPrAn) R package for the study of macromolecular complexes.

Many cellular processes involve the participation of large macromolecular assemblies. Understanding their function requires methods allowing to study their dynamic and mechanistic properties. Here we present a method for quantitative analysis of native protein or ribonucleoprotein complexes by mass spectrometry following their separation by density - qDGMS. Mass spectrometric quantitation is enabled through stable isotope labelling with amino acids in cell culture (SILAC). We provide a complete guide, from experimental design to preparation of publication-ready figures, using a purposely-developed R package - ComPrAn. As specific examples, we present the use of sucrose density gradients to inspect the assembly and dynamics of the human mitochondrial ribosome (mitoribosome), its interacting proteins, the small subunit of the cytoplasmic ribosome, cytoplasmic aminoacyl-tRNA synthetase complex and the mitochondrial PDH complex. ComPrAn provides tools for analysis of peptide-level data as well as normalization and clustering tools for protein-level data, dedicated visualization functions and graphical user interface. Although, it has been developed for the analysis of qDGMS samples, it can also be used for other proteomics experiments that involve 2-state labelled samples separated into fractions. We show that qDGMS and ComPrAn can be used to study macromolecular complexes in their native state, accounting for the dynamics inherent to biological systems and benefiting from its proteome-wide quantitative and qualitative capability.

Aberrant splicing in neuroblastoma generates RNA-fusion transcripts and provides vulnerability to spliceosome inhibitors.

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.

Mitoribosome Profiling from Human Cell Culture: A High Resolution View of Mitochondrial Translation.

Ribosome profiling (Ribo-Seq) is a technique that allows genome-wide, quantitative analysis of translation. In recent years, it has found multiple applications in studies of translation in diverse organisms, tracking protein synthesis with single codon resolution. Traditional protocols applied for generating Ribo-Seq libraries from mammalian cell cultures are not suitable to study mitochondrial translation due to differences between eukaryotic cytosolic and mitochondrial ribosomes. Here, we present an adapted protocol enriching for mitoribosome footprints. In addition, we describe the preparation of small RNA sequencing libraries from the resultant mitochondrial ribosomal protected fragments (mtRPFs).