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.

Decoding the Enigma: Translation Termination in Human Mitochondria.

Mitochondrial translation is a complex process responsible for the synthesis of essential proteins involved in oxidative phosphorylation, a fundamental pathway for cellular energy production. Central to this process is the termination phase, where dedicated factors play a pivotal role in ensuring accurate and timely protein production. This review provides a comprehensive overview of the current understanding of translation termination in human mitochondria, emphasizing structural features and molecular functions of two mitochondrial termination factors mtRF1 and mtRF1a.

The human mitochondrial translation factor TACO1 alleviates mitoribosome stalling at polyproline stretches.

The prokaryotic translation elongation factor P (EF-P) and the eukaryotic/archaeal counterparts eIF5A/aIF5A are proteins that serve a crucial role in mitigating ribosomal stalling during the translation of specific sequences, notably those containing consecutive proline residues (1,2). Although mitochondrial DNA-encoded proteins synthesized by mitochondrial ribosomes also contain polyproline stretches, an EF-P/eIF5A mitochondrial counterpart remains unidentified. Here, we show that the missing factor is TACO1, a protein causative of a juvenile form of neurodegenerative Leigh's syndrome associated with cytochrome c oxidase deficiency, until now believed to be a translational activator of COX1 mRNA. By using a combination of metabolic labeling, puromycin release and mitoribosome profiling experiments, we show that TACO1 is required for the rapid synthesis of the polyproline-rich COX1 and COX3 cytochrome c oxidase subunits, while its requirement is negligible for other mitochondrial DNA-encoded proteins. In agreement with a role in translation efficiency regulation, we show that TACO1 cooperates with the N-terminal extension of the large ribosomal subunit bL27m to provide stability to the peptidyl-transferase center during elongation. This study illuminates the translation elongation dynamics within human mitochondria, a TACO1-mediated biological mechanism in place to mitigate mitoribosome stalling at polyproline stretches during protein synthesis, and the pathological implications of its malfunction.

Influence of chain length and branching on poly(ADP-ribose)-protein interactions.

Hundreds of proteins interact with poly(ADP-ribose) (PAR) via multiple PAR interaction motifs, thereby regulating their physico-chemical properties, sub-cellular localizations, enzymatic activities, or protein stability. Here, we present a targeted approach based on fluorescence correlation spectroscopy (FCS) to characterize potential structure-specific interactions of PAR molecules of defined chain length and branching with three prime PAR-binding proteins, the tumor suppressor protein p53, histone H1, and the histone chaperone APLF. Our study reveals complex and structure-specific PAR-protein interactions. Quantitative Kd values were determined and binding affinities for all three proteins were shown to be in the nanomolar range. We report PAR chain length dependent binding of p53 and H1, yet chain length independent binding of APLF. For all three PAR binders, we found a preference for linear over hyperbranched PAR. Importantly, protein- and PAR-structure-specific binding modes were revealed. Thus, while the H1-PAR interaction occurred largely on a bi-molecular 1:1 basis, p53-and potentially also APLF-can form complex multivalent PAR-protein structures. In conclusion, our study gives detailed and quantitative insight into PAR-protein interactions in a solution-based setting at near physiological buffer conditions. The results support the notion of protein and PAR-structure-specific binding modes that have evolved to fit the purpose of the respective biochemical functions and biological contexts.

Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly.

Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.

PARP1 catalytic variants reveal branching and chain length-specific functions of poly(ADP-ribose) in cellular physiology and stress response.

Poly(ADP-ribosyl)ation regulates numerous cellular processes like genome maintenance and cell death, thus providing protective functions but also contributing to several pathological conditions. Poly(ADP-ribose) (PAR) molecules exhibit a remarkable heterogeneity in chain lengths and branching frequencies, but the biological significance of this is basically unknown. To unravel structure-specific functions of PAR, we used PARP1 mutants producing PAR of different qualities, i.e. short and hypobranched (PARP1\G972R), short and moderately hyperbranched (PARP1\Y986S), or strongly hyperbranched PAR (PARP1\Y986H). By reconstituting HeLa PARP1 knockout cells, we demonstrate that PARP1\G972R negatively affects cellular endpoints, such as viability, cell cycle progression and genotoxic stress resistance. In contrast, PARP1\Y986S elicits only mild effects, suggesting that PAR branching compensates for short polymer length. Interestingly, PARP1\Y986H exhibits moderate beneficial effects on cell physiology. Furthermore, different PARP1 mutants have distinct effects on molecular processes, such as gene expression and protein localization dynamics of PARP1 itself, and of its downstream factor XRCC1. Finally, the biological relevance of PAR branching is emphasized by the fact that branching frequencies vary considerably during different phases of the DNA damage-induced PARylation reaction and between different mouse tissues. Taken together, this study reveals that PAR branching and chain length essentially affect cellular functions, which further supports the notion of a 'PAR code'.

Interactions of p53 with poly(ADP-ribose) and DNA induce distinct changes in protein structure as revealed by ATR-FTIR spectroscopy.

Due to multiple domains and in part intrinsically disordered regions, structural analyses of p53 remain a challenging task, particularly in complex with DNA and other macromolecules. Here, we applied a novel attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic approach to investigate changes in secondary structure of full-length p53 induced by non-covalent interactions with DNA and poly(ADP-ribose) (PAR). To validate our approach, we confirmed a positive regulatory function of p53's C-terminal domain (CTD) with regard to sequence-specific DNA binding and verified that the CTD mediates p53-PAR interaction. Further, we demonstrate that DNA and PAR interactions result in distinct structural changes of p53, indicating specific binding mechanisms via different domains. A time-dependent analysis of the interplay of DNA and PAR binding to p53 revealed that PAR represents p53's preferred binding partner, which efficiently controls p53-DNA interaction. Moreover, we provide infrared spectroscopic data on PAR pointing to the absence of regular secondary structural elements. Finally, temperature-induced melting experiments via CD spectroscopy show that DNA binding stabilizes the structure of p53, while PAR binding can shift the irreversible formation of insoluble p53 aggregates to higher temperatures. In conclusion, this study provides detailed insights into the dynamic interplay of p53 binding to DNA and PAR at a formerly inaccessible molecular level.

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.

The C-terminal domain of p53 orchestrates the interplay between non-covalent and covalent poly(ADP-ribosyl)ation of p53 by PARP1.

The post-translational modification poly(ADP-ribosyl)ation (PARylation) plays key roles in genome maintenance and transcription. Both non-covalent poly(ADP-ribose) binding and covalent PARylation control protein functions, however, it is unknown how the two modes of modification crosstalk mechanistically. Employing the tumor suppressor p53 as a model substrate, this study provides detailed insights into the interplay between non-covalent and covalent PARylation and unravels its functional significance in the regulation of p53. We reveal that the multifunctional C-terminal domain (CTD) of p53 acts as the central hub in the PARylation-dependent regulation of p53. Specifically, p53 bound to auto-PARylated PARP1 via highly specific non-covalent PAR-CTD interaction, which conveyed target specificity for its covalent PARylation by PARP1. Strikingly, fusing the p53-CTD to a protein that is normally not PARylated, renders this a target for covalent PARylation as well. Functional studies revealed that the p53-PAR interaction had substantial implications on molecular and cellular levels. Thus, PAR significantly influenced the complex p53-DNA binding properties and controlled p53 functions, with major implications on the p53-dependent interactome, transcription, and replication-associated recombination. Remarkably, this mechanism potentially also applies to other PARylation targets, since a bioinformatics analysis revealed that CTD-like regions are highly enriched in the PARylated proteome.

Kinetics of poly(ADP-ribosyl)ation, but not PARP1 itself, determines the cell fate in response to DNA damage in vitro and in vivo.

One of the fastest cellular responses to genotoxic stress is the formation of poly(ADP-ribose) polymers (PAR) by poly(ADP-ribose)polymerase 1 (PARP1, or ARTD1). PARP1 and its enzymatic product PAR regulate diverse biological processes, such as DNA repair, chromatin remodeling, transcription and cell death. However, the inter-dependent function of the PARP1 protein and its enzymatic activity clouds the mechanism underlying the biological response. We generated a PARP1 knock-in mouse model carrying a point mutation in the catalytic domain of PARP1 (D993A), which impairs the kinetics of the PARP1 activity and the PAR chain complexity in vitro and in vivo, designated as hypo-PARylation. PARP1D993A/D993A mice and cells are viable and show no obvious abnormalities. Despite a mild defect in base excision repair (BER), this hypo-PARylation compromises the DNA damage response during DNA replication, leading to cell death or senescence. Strikingly, PARP1D993A/D993A mice are hypersensitive to alkylation in vivo, phenocopying the phenotype of PARP1 knockout mice. Our study thus unravels a novel regulatory mechanism, which could not be revealed by classical loss-of-function studies, on how PAR homeostasis, but not the PARP1 protein, protects cells and organisms from acute DNA damage.

Staphylococcus epidermidis bacteriocin A37 kills natural competitors with a unique mechanism of action.

Many bacteria produce antimicrobial compounds such as lantibiotics to gain advantage in the competitive natural environments of microbiomes. Epilancins constitute an until now underexplored family of lantibiotics with an unknown ecological role and unresolved mode of action. We discovered production of an epilancin in the nasal isolate Staphylococcus epidermidis A37. Using bioinformatic tools, we found that epilancins are frequently encoded within staphylococcal genomes, highlighting their ecological relevance. We demonstrate that production of epilancin A37 contributes to Staphylococcus epidermidis competition specifically against natural corynebacterial competitors. Combining microbiological approaches with quantitative in vivo and in vitro fluorescence microscopy and cryo-electron tomography, we show that A37 enters the corynebacterial cytoplasm through a partially transmembrane-potential-driven uptake without impairing the cell membrane function. Upon intracellular aggregation, A37 induces the formation of intracellular membrane vesicles, which are heavily loaded with the compound and are essential for the antibacterial activity of the epilancin. Our work sheds light on the ecological role of epilancins for staphylococci mediated by a mode of action previously unknown for lantibiotics.

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.

The role of effect-based methods to address water quality monitoring in South Africa: a developing country's struggle.

Water is an important resource, and it is a worldwide struggle to provide water of good quality to the whole population. Despite good governing laws and guidelines set in place to help protect the water resources and ensure it is of good quality for various consumers, the water quality in South Africa is worsening due to lack of management. The deteriorating infrastructure is becoming progressively worse, due to corruption and insufficient funds. The ever-increasing number of toxicants, as well as the identification of emerging chemicals of concern, are also challenges South Africa is facing. Chemical analysis cannot determine the total biological effect of a mixture of chemical compounds, but this shortcoming can be addressed by adding effect-based methods (EBMs) to water quality monitoring programmes. In this paper, the current status of water quality monitoring in South Africa is discussed, as well as the capacity of the country to add EBMs to its water quality monitoring programmes to protect and improve human and animal life. Created in Biorender.com.

Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time.

Ribosomal biogenesis has been studied by biochemical, genetic and electron microscopic approaches, but live cell data on the in vivo kinetics are still missing. Here we analyse the export kinetics of the large ribosomal subunit (pre-60S particle) through single NPCs in human cells. We established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label pre-60S particles and NPCs, respectively. By combining single molecule tracking and super resolution confocal microscopy we visualize the dynamics of single pre-60S particles during export through single NPCs. For export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export has a single rate limiting step and a duration of ∼24 milliseconds. Only about 1/3 of attempted export events are successful. Our results show that the mass flux through a single NPC can reach up to ~125 MDa·s-1 in vivo.

Real-time monitoring of PARP1-dependent PARylation by ATR-FTIR spectroscopy.

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD+ substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3' phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD+ and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.