bims-mitran 2023-05-14 papers

bims-mitran

Biomed News

on Mitochondrial translation

Issue of 2023–05–14
twenty papers selected by
Andreas Kohler, University of Graz

Sucrose Gradient Analysis of Human Mitochondrial Ribosomes and RNA.
Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation.
Discovery of Mitochondrial Ribosomes.
Assembly of the Mitochondrial Translation Initiation Complex.
Insights into mitoribosomal biogenesis from recent structural studies.
Translation in Mitochondrial Ribosomes.
Reconstitution of Mammalian Mitochondrial Translation System Capable of Long Polypeptide Synthesis.
Human Mitoribosome Profiling: A Re-engineered Approach Tailored to Study Mitochondrial Translation.
Metabolic Labeling of Mitochondrial Translation Products in Whole Cells and Isolated Organelles.
Mitoribosome Biogenesis.
The ARG8m Reporter for the Study of Yeast Mitochondrial Translation.
Four-Color STED Super-Resolution RNA Fluorescent In Situ Hybridization and Immunocytochemistry to Visualize Mitochondrial mRNAs in Context with Mitochondrial Ribosomes.
Digital RNase Footprinting of RNA-Protein Complexes and Ribosomes in Mitochondria.
Cryo-EM for Structure Determination of Mitochondrial Ribosome Samples.
Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway.
Evolution: Mitochondrial Ribosomes Across Species.
Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation.
Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.
Rapid Cryopurification of the Yeast Mitochondrial Ribosome.
Newly imported proteins in mitochondria are particularly sensitive to aggregation.

Methods Mol Biol. 2023 ;2661 101-117

Sucrose Gradient Analysis of Human Mitochondrial Ribosomes and RNA.

Kah Ying Ng, Brendan J Battersby.

  Faithful expression of the mitochondrial genome is required for the synthesis of the oxidative phosphorylation complexes and cell fitness. In humans, mitochondrial DNA (mtDNA) encodes 13 essential subunits of four oxidative phosphorylation complexes along with tRNAs and rRNAs needed for the translation of these proteins. Protein synthesis occurs on unique ribosomes within the organelle. Over the last decade, the revolution in genetic diagnostics has identified disruptions to the faithful synthesis of these 13 mitochondrial proteins as the largest group of inherited human mitochondrial pathologies. All of the molecular steps required for mitochondrial protein synthesis can be affected, from the genome to protein, including cotranslational quality control. Here, we describe methodologies for the biochemical separation of mitochondrial ribosomes from cultured human cells for RNA and protein analysis. Our method has been optimized to facilitate analysis for low-level sample material and thus does not require prior organelle enrichment.

Keywords:  Human disease; Mitochondria; Mitochondrial disease; RNA; Ribosomes; Sucrose gradient

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_7
Methods Mol Biol. 2023 ;2661 119-132

Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation.

Andreas Aufschnaiter, Andreas Carlström, Martin Ott.

  Mitochondrial protein biosynthesis is maintained by an interplay between the mitochondrial ribosome (mitoribosome) and a large set of protein interaction partners. This interactome regulates a diverse set of functions, including mitochondrial gene expression, translation, protein quality control, and respiratory chain assembly. Hence, robust methods to biochemically and structurally analyze this molecular machinery are required to understand the sophisticated regulation of mitochondrial protein biosynthesis. In this chapter, we present detailed protocols for immunoprecipitation, sucrose cushions, and linear sucrose gradients to purify and analyze mitoribosomes and their interaction partners.

Keywords:  Immunoprecipitation; Mitochondria; Mitoribosome; Sucrose cushion; Sucrose gradient; Yeast

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_8
Methods Mol Biol. 2023 ;2661 3-5

Discovery of Mitochondrial Ribosomes.

Thomas W O'Brien.

  In this introductory chapter, I will briefly describe how I came to discover the mammalian mitoribosome and will add a few notes on my contribution to the field.

Keywords:  Human MRP genes; Human ribosome purification; Mitochondrial disease; Mitochondrial ribosomal proteins; Mitochondrial translation

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_1
Methods Mol Biol. 2023 ;2661 217-232

Assembly of the Mitochondrial Translation Initiation Complex.

Cristina Remes, Minh Duc Nguyen, Henrik Spahr, Martin Ng, Clark Fritsch, Arpan Bhattacharya, Hong Li, Barry Cooperman, Joanna Rorbach.

  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.

Keywords:  Mitoribosome; Translation; Translation factors; Translation initiation; tRNA purification

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_13
Trends Biochem Sci. 2023 May 09. pii: S0968-0004(23)00086-5. [Epub ahead of print]

Insights into mitoribosomal biogenesis from recent structural studies.

Anas Khawaja, Miriam Cipullo, Annika Krüger, Joanna Rorbach.

  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.

Keywords:  assembly factors; mitochondrial ribosome; mitoribosome assembly; ribosomal RNA

DOI:  https://doi.org/10.1016/j.tibs.2023.04.002
Methods Mol Biol. 2023 ;2661 53-72

Translation in Mitochondrial Ribosomes.

Zofia M Chrzanowska-Lightowlers, Robert N Lightowlers.

  Mitochondrial protein synthesis is essential for the life of aerobic eukaryotes. Without it, oxidative phosphorylation cannot be coupled. Evolution has shaped a battery of factors and machinery that are key to production of just a handful of critical proteins. In this general concept chapter, we attempt to briefly summarize our current knowledge of the overall process in mitochondria from a variety of species, breaking this down to the four parts of translation: initiation, elongation, termination, and recycling. Where appropriate, we highlight differences between species and emphasize gaps in our understanding. Excitingly, with the current revolution in cryoelectron microscopy and mitochondrial genome editing, it is highly likely that many of these gaps will be resolved in the near future. However, the absence of a faithful in vitro reconstituted system to study mitochondrial translation is still problematic.

Keywords:  Elongation; Initiation; Mitochondria; Mitoribosomes; Protein synthesis; Recycling; Termination; Translation; mt-mRNAs

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_4
Methods Mol Biol. 2023 ;2661 233-255

Reconstitution of Mammalian Mitochondrial Translation System Capable of Long Polypeptide Synthesis.

Muhoon Lee, Nono Takeuchi-Tomita.

  Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. Here, we describe the in vitro reconstitution of the mammalian mitochondrial translation system, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a heterologous yeast tRNA mixture. The system is capable of translating leaderless mRNAs encoding model proteins, such as nanoluciferase with a molecular weight of 19 kDa, and is readily applicable for in vitro evaluations of mRNAs and nascent peptide chain sequences, as well as factors and small molecules that affect mitochondrial translation.

Keywords:  55S ribosome; In vitro translation; Leaderless mRNA; Mammalian mitochondria; Reconstituted translation system

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_14
Methods Mol Biol. 2023 ;2661 257-280

Human Mitoribosome Profiling: A Re-engineered Approach Tailored to Study Mitochondrial Translation.

Iliana Soto, Mary Couvillion, L Stirling Churchman.

  To understand the human mitochondrial translation process, tools are required to dissect this system at a global scale. The mechanisms and regulation of translation in mitochondria are different from those in the cytosol, and mitochondrial ribosomes have distinct biochemical properties. In this chapter, we describe in detail the modifications we have made to the ribosome profiling approach to adapt it to the unique characteristics of the human mitochondrial ribosome. This approach maximizes the fraction of mitochondrial ribosomes recovered, providing a snapshot of the mitochondrial translation landscape with minimal bias. We also describe the use of mouse lysate as an internal spike-in control for normalization, allowing quantification of global changes in translation across samples. Finally, we outline the bioinformatic pipelines to process the raw reads and identify mitoribosome A sites in the absence of untranslated regions flanking open reading frames. This method offers a subcodon-resolution time-sensitive global approach to explore the mitochondrial translation process in human cells.

Keywords:  Human mitochondrial translation; Mitochondrial ribosome profiling

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_15
Methods Mol Biol. 2023 ;2661 193-215

Metabolic Labeling of Mitochondrial Translation Products in Whole Cells and Isolated Organelles.

Priyanka Maiti, Flavia Fontanesi.

  Mitochondria retain their own genome and translational apparatus that is highly specialized in the synthesis of a handful of proteins, essential components of the oxidative phosphorylation system. During evolution, the players and mechanisms involved in mitochondrial translation have acquired some unique features, which we have only partially disclosed. The study of the mitochondrial translation process has been historically hampered by the lack of an in vitro translational system and has largely relied on the analysis of the incorporation rate of radiolabeled amino acids into mitochondrial proteins in cellulo or in organello. In this chapter, we describe methods to monitor mitochondrial translation by labeling newly synthesized mitochondrial polypeptides with [S35]-methionine in either yeast or mammalian whole cells or isolated mitochondria.

Keywords:  Human cells; Mitochondrial translation; Newly synthesized polypeptides; Protein synthesis; Pulse-chase labeling; Yeast; [S35]-methionine

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_12
Methods Mol Biol. 2023 ;2661 23-51

Mitoribosome Biogenesis.

J Conor Moran, Samuel Del'Olio, Austin Choi, Hui Zhong, Antoni Barrientos.

  Mitoribosome biogenesis is a complex and energetically costly process that involves RNA elements encoded in the mitochondrial genome and mitoribosomal proteins most frequently encoded in the nuclear genome. The process is catalyzed by extra-ribosomal proteins, nucleus-encoded assembly factors that act in all stages of the assembly process to coordinate the processing and maturation of ribosomal RNAs with the hierarchical association of ribosomal proteins. Biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided hints regarding their assembly. In this general concept chapter, we will briefly describe the current knowledge, mainly regarding the mammalian mitoribosome biogenesis pathway and factors involved, and will emphasize the biological sources and approaches that have been applied to advance the field.

Keywords:  Mitochondrial disease; Mitochondrial ribosome; Mitochondrial translation; Mitoribosome assembly; OXPHOS deficiency

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_3
Methods Mol Biol. 2023 ;2661 281-301

The ARG8m Reporter for the Study of Yeast Mitochondrial Translation.

Daniel Flores-Mireles, Yolanda Camacho-Villasana, Xochitl Pérez-Martínez.

  Mitochondrial translation is an intricate process involving both general and mRNA-specific factors. In addition, in the yeast Saccharomyces cerevisiae, translation of mitochondrial mRNAs is coupled to assembly of nascent polypeptides into the membrane. ARG8m is a reporter gene widely used to study the mechanisms of yeast mitochondrial translation. This reporter is a recodified gene that uses the mitochondrial genetic code and is inserted at the desired locus in the mitochondrial genome. After deletion of the endogenous nuclear gene, this reporter produces Arg8, an enzyme necessary for arginine biosynthesis. Since Arg8 is a soluble protein with no relation to oxidative phosphorylation, it is a reliable reporter to study mitochondrial mRNAs translation and dissect translation form assembly processes. In this chapter, we explain how to insert the ARG8m reporter in the desired spot in the mitochondrial DNA, how to analyze Arg8 synthesis inside mitochondria, and how to follow steady-state levels of the protein. We also explain how to use it to find spontaneous suppressors of translation defects.

Keywords:  ARG8m; ATP synthase; Cytochrome c oxidase; Mitochondria; Mitochondrial DNA; Respiratory complexes; Suppressor; Translation; Yeast; bc1 complex

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_16
Methods Mol Biol. 2023 ;2661 303-316

Four-Color STED Super-Resolution RNA Fluorescent In Situ Hybridization and Immunocytochemistry to Visualize Mitochondrial mRNAs in Context with Mitochondrial Ribosomes.

Christin A Albus, Rolando Berlinguer-Palmini, Zofia M Chrzanowska-Lightowlers, Robert N Lightowlers.

  High-resolution imaging has enabled scientists to explore the mitochondrial network at remarkable resolution. This has been exploited to help increase our knowledge of how mitochondrial gene expression is compartmentalized in cultured cells. Here, we provide detailed methodology to simultaneously visualize up to four components including mtDNA-encoded transcripts, submitochondrial marker proteins, mitoribosomal subunits, or core members of the translational apparatus using STED super-resolution nanoscopy.

Keywords:  Mitochondrial mRNA; RNA in situ hybridization; STED super-resolution; Super-resolution nanoscopy

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_17
Methods Mol Biol. 2023 ;2661 317-328

Digital RNase Footprinting of RNA-Protein Complexes and Ribosomes in Mitochondria.

Danielle L Rudler, Stefan J Siira, Oliver Rackham, Aleksandra Filipovska.

  RNA-binding proteins and mitochondrial ribosomes have been found to be linchpins of mitochondrial gene expression in health and disease. The expanding repertoire of proteins that bind and regulate the mitochondrial transcriptome has necessitated the development of new tools and methods to examine their molecular functions. Next-generation sequencing technologies have advanced the RNA biology field through application of high-throughput methods to study RNA-protein interactions. Here we describe a digital RNase footprinting method to analyze protein and ribosome interactions with mitochondrially encoded transcripts that provides insight into their mechanisms and minimal binding sites. We provide details on RNase digestion and next-generation sequencing, along with computational analyses and visualization of the binding targets within the mitochondrial transcriptome.

Keywords:  Bioinformatics; Footprinting; Mitoribosome; RNA-Seq; RNA-binding proteins; mtDNA

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_18
Methods Mol Biol. 2023 ;2661 89-100

Cryo-EM for Structure Determination of Mitochondrial Ribosome Samples.

Hauke S Hillen.

  Single-particle cryoelectron microscopy (cryo-EM) allows structure determination of large macromolecular complexes from conformationally and compositionally heterogeneous mixtures of particles. This technique has been used to reveal the architecture of the mitochondrial ribosome and to visualize transient states that occur during the translation cycle or during mitoribosome biogenesis. Here, we outline an exemplary workflow for the analysis of single-particle cryo-EM data of human mitoribosome samples. In addition, we provide an example dataset which can be used for training purposes alongside the protocol.

Keywords:  Mitochondrial ribosome; image processing; sample preparation; single-particle cryo-EM

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_6
Methods Mol Biol. 2023 ;2661 163-191

Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway.

Samuel Del'Olio, Antoni Barrientos.

  Studies of yeast mitoribosome assembly have been historically hampered by the difficulty of generating mitoribosome protein-coding gene deletion strains with a stable mitochondrial genome. The identification of mitochondrial DNA-stabilizing approaches allows for the generation of a complete set of yeast deletion strains covering all mitoribosome proteins and known assembly factors. These strains can be used to analyze the integrity and assembly state of mitoribosomes by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts, coupled to mass spectrometry analysis of mitoribosome composition. Subsequent hierarchical cluster analysis of mitoribosome subassemblies accumulated in mutant strains reveals details regarding the order of protein association during the mitoribosome biogenetic process. These strains also allow the expression of truncated protein variants to probe the role of mitochondrion-specific protein extensions, the relevance of protein cofactors, or the importance of RNA-protein interactions in functional sites of the mitoribosome. In this chapter, we will detail the methodology involved in these studies.

Keywords:  Clustering analysis; Gradient fractionation; Immunoblotting; Mass spectrometry; Mitochondrial ribosome; Mitoribosome assembly intermediate; Mitoribosome profile; Sucrose gradient; Yeast mitoribosome gene deletion strain

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_11
Methods Mol Biol. 2023 ;2661 7-21

Evolution: Mitochondrial Ribosomes Across Species.

Rajendra K Agrawal, Soneya Majumdar.

  The ribosome is among the most complex and ancient cellular macromolecular assemblies that plays a central role in protein biosynthesis in all living cells. Its function of translation of genetic information encoded in messenger RNA into protein molecules also extends to subcellular compartments in eukaryotic cells such as apicoplasts, chloroplasts, and mitochondria. The origin of mitochondria is primarily attributed to an early endosymbiotic event between an alpha-proteobacterium and a primitive (archaeal) eukaryotic cell. The timeline of mitochondrial acquisition, the nature of the host, and their diversification have been studied in great detail and are continually being revised as more genomic and structural data emerge. Recent advancements in high-resolution cryo-EM structure determination have provided architectural details of mitochondrial ribosomes (mitoribosomes) from various species, revealing unprecedented diversifications among them. These structures provide novel insights into the evolution of mitoribosomal structure and function. Here, we present a brief overview of the existing mitoribosomal structures in the context of the eukaryotic evolution tree showing their diversification from their last common ancestor.

Keywords:  Cryo-EM structures; Evolution; MRPs; Mitochondrial rRNAs; Mitochondrial ribosomes

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_2
Methods Mol Biol. 2023 ;2661 75-88

Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation.

Lena Thärichen, Robert Englmeier, Friedrich Förster.

  Cryoelectron tomography is a method to image biological samples three-dimensionally at molecular resolution. This modality provides insights into intracellular processes in their physiological settings. Obtaining a high-quality sample for cryoelectron tomography on mitochondria, however, can be challenging. In this chapter, we describe the crucial steps from sample preparation to data acquisition enabling studies of mitochondrial translation in situ by cryoelectron tomography. We provide detailed protocols for yeast and human mitochondria preparations yielding a high concentration of intact mitochondrial vesicles on cryo-EM grids. In addition, we describe a workflow for particle identification and spatial mapping in context of the organelle.

Keywords:  Cryoelectron tomography; Mitochondria; Ribosome; Translation

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_5
Methods Mol Biol. 2023 ;2661 143-161

Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.

Lea Bertgen, Tamara Flohr, Johannes M Herrmann.

  The biogenesis of mitoribosomes is an intricate process that relies on the coordinated synthesis of nuclear-encoded mitoribosomal proteins (MRPs) in the cytosol, their translocation across mitochondrial membranes, the transcription of rRNA molecules in the matrix as well as the assembly of the roughly 80 different constituents of the mitoribosome. Numerous chaperones, translocases, processing peptidases, and assembly factors of the cytosol and in mitochondria support this complex reaction. The budding yeast Saccharomyces cerevisiae served as a powerful model organism to unravel the different steps by which MRPs are imported into mitochondria, fold into their native structures, and assemble into functional ribosomes.In this chapter, we provide established protocols to study these different processes experimentally. In particular, we describe methods to purify mitochondria from yeast cells, to import radiolabeled MRPs into isolated mitochondria, and to elucidate the assembly reaction of MRPs by immunoprecipitation. These protocols and the list of dos and don'ts will enable beginners and experienced scientists to study the import and assembly of MRPs.

Keywords:  Immunoprecipitation; Isolation of mitochondria; Mitochondrial protein import; Mitoribosomal protein (MRP); Sample preparation for mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_10
Methods Mol Biol. 2023 ;2661 133-141

Rapid Cryopurification of the Yeast Mitochondrial Ribosome.

Hong Weng Pang, Antoni Barrientos.

  Cryogenic milling, or cryomilling, involves the use of liquid nitrogen to lower the temperature of the biological material and/or the milling process. When applied to the study of subcellular or suborganellar structures and processes, it allows for their rapid extraction from whole cells frozen in the physiological state of choice. This approach has proven to be useful for the study of yeast mitochondrial ribosomes. Following cryomilling of 100 mL of yeast culture, conveniently tagged mitochondrial ribosomes can be immunoprecipitated and purified in native conditions. These ribosomes are suitable for the application of downstream approaches. These include mitoribosome profiling to analyze the mitochondrial translatome or mass spectrometry analyses to assess the mitoribosome proteome in normal growth conditions or under stress, as described in this method.

Keywords:  Cryomilling; Immunoblotting; Immunoprecipitation; Mass spectrometry; Yeast mitoribosome

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_9
Acta Physiol (Oxf). 2023 May 12. e13985

Newly imported proteins in mitochondria are particularly sensitive to aggregation.

Carmela Vazquez-Calvo, Verena Kohler, Johanna L Höög, Sabrina Büttner, Martin Ott.

   AIM: A functional proteome is essential for life and maintained by protein quality control (PQC) systems in the cytosol and organelles. Protein aggregation is an indicator of a decline of PQC linked to aging and disease. Mitochondrial PQC is critical to maintain mitochondrial function and thus cellular fitness. How mitochondria handle aggregated proteins is not well understood. Here we tested how the metabolic status impacts on formation and clearance of aggregates within yeast mitochondria and assessed which proteins are particularly sensitive to denaturation.
METHODS: Confocal microscopy, electron microscopy, immunoblotting and genetics were applied to assess mitochondrial aggregate handling in response to heat shock and ethanol, using the mitochondrial disaggregase Hsp78 as a marker for protein aggregates.
RESULTS: We show that aggregates formed upon heat or ethanol stress with different dynamics depending on the metabolic state. While fermenting cells displayed numerous small aggregates that coalesced into one large foci that was resistant to clearance, respiring cells showed less aggregates and cleared these aggregates more efficiently. Acute inhibition of mitochondrial translation had no effect, while preventing protein import into mitochondria by inhibition of cytosolic translation prevented aggregate formation.
CONCLUSION: Collectively, our data show that the metabolic state of the cells impacts the dynamics of aggregate formation and clearance, and that mainly newly imported and not yet assembled proteins are prone to form aggregates. Because mitochondrial functionality is crucial for cellular metabolism, these results highlight the importance of efficient protein biogenesis to maintain the mitochondrial proteome operational during metabolic adaptations and cellular stress.

Keywords:  Ageing; Aggregates; Cellular stress; Hsp78; Metabolism; Mitochondria; Protein quality control; Proteostasis

DOI:  https://doi.org/10.1111/apha.13985