Discovery of Mitochondrial Ribosomes.

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.

Evolution: Mitochondrial Ribosomes Across Species.

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.

Rapid Cryopurification of the Yeast Mitochondrial Ribosome.

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.

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

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.

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

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.

Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.

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.

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

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.

Global Assessment of Protein Translation in Mammalian Cells Using Polysome Fractionation.

A character of active protein translation is formation of multiple ribosomes, or polysomes, on translating mRNAs. Polysome intensity reflects global cellular translation activity and can be assessed after biochemical fractionations of polysomes. Polysome fractionation begins with immobilizing ribosomes on mRNAs using inhibitors of translation elongation, for example, cycloheximide. Nuclei-free cell lysates are then isolated and layered on the top of a sucrose gradient for ultracentrifugation to separate ribosomal subunits, monosome, and multiple fractions of polysomes by their different sedimentation rates along the sucrose gradient. A density gradient fractionation system including a spectrophotometer reads the RNA absorbance of the flowed gradient and generates the fractions. These fractions can be subjected to further RNA and protein analyses, for example, polysome profiling and mass spectrometry. Here, we present a detailed protocol of polysome fractionation for mammalian cells.

Ribosomal Profiling by Gradient Fractionation of Cell Lysates.

Ribosomal profiling is a widely used technique for deep sequencing of ribosome-protected mRNA and for measuring ribosome status in cells. It is a powerful method that is typically employed for monitoring and measuring protein translation status and ribosome activity. Also, it has been used for monitoring the ribosomal stress-responsive events in the ribosome activity. Furthermore, this approach enables understanding of translational regulation, which is invisible in most proteomic approaches. Moreover, this method is known as an important approach for biological discovery such as identification of translation products. Hence, this methodology will be useful for studying cellular events engaging in ribosome assembly, ribosome biogenesis, ribosome activity, translation during the cell cycle, cell proliferation, and growth as well as the ribosomal stress response in mammalian cells.

SnapShot: Eukaryotic ribosome biogenesis I.

Ribosome production is vital for every cell, and failure causes human diseases. It is driven by ∼200 assembly factors functioning along an ordered pathway from the nucleolus to the cytoplasm. Structural snapshots of biogenesis intermediates from the earliest 90S pre-ribosomes to mature 40S subunits unravel the mechanisms of small ribosome synthesis. To view this SnapShot, open or download the PDF.

Fix it, don't trash it: Ribosome maintenance by chaperone-mediated repair of damaged subunits.

Acute stressors or normal cellular function may result in ribosomal protein damage, which threatens the functional ribosome pool and translation. In this issue, Yang et al.1 show that chaperones can extract damaged ribosomal proteins and replace them with newly synthesized versions to repair mature ribosomes.

Decoding and Recoding of mRNA Sequences by the Ribosome.

Faithful translation of messenger RNA (mRNA) into protein is essential to maintain protein homeostasis in the cell. Spontaneous translation errors are very rare due to stringent selection of cognate aminoacyl transfer RNAs (tRNAs) and the tight control of the mRNA reading frame by the ribosome. Recoding events, such as stop codon readthrough, frameshifting, and translational bypassing, reprogram the ribosome to make intentional mistakes and produce alternative proteins from the same mRNA. The hallmark of recoding is the change of ribosome dynamics. The signals for recoding are built into the mRNA, but their reading depends on the genetic makeup of the cell, resulting in cell-specific changes in expression programs. In this review, I discuss the mechanisms of canonical decoding and tRNA-mRNA translocation; describe alternative pathways leading to recoding; and identify the links among mRNA signals, ribosome dynamics, and recoding.

Therapeutic targeting of CNBP phase separation inhibits ribosome biogenesis and neuroblastoma progression via modulating SWI/SNF complex activity.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial malignancy in childhood; however, the mechanisms underlying its aggressive characteristics still remain elusive. METHODS: Integrative data analysis was performed to reveal tumour-driving transcriptional regulators. Co-immunoprecipitation and mass spectrometry assays were applied for protein interaction studies. Real-time reverse transcription-polymerase chain reaction, western blotting, sequential chromatin immunoprecipitation and dual-luciferase reporter assays were carried out to explore gene expression regulation. The biological characteristics of NB cell lines were examined via gain- and loss-of-function assays. For survival analysis, the Cox regression model and log-rank tests were used. RESULTS: Cellular nucleic acid-binding protein (CNBP) was found to be an independent factor affecting NB outcome, which exerted oncogenic roles in ribosome biogenesis, tumourigenesis and aggressiveness. Mechanistically, karyopherin subunit beta 1 (KPNB1) was responsible for nuclear transport of CNBP, whereas liquid condensates of CNBP repressed the activity of switch/sucrose-nonfermentable (SWI/SNF) core subunits (SMARCC2/SMARCC1/SMARCA4) via interaction with SMARCC2, leading to alternatively increased activity of SMARCC1/SMARCA4 binary complex in facilitating gene expression essential for 18S ribosomal RNA (rRNA) processing in tumour cells, extracellular vesicle-mediated delivery of 18S rRNA and subsequent M2 macrophage polarisation. A cell-penetrating peptide blocking phase separation and interaction of CNBP with SMARCC2 inhibited ribosome biogenesis and NB progression. High KPNB1, CNBP, SMARCC1 or SMARCA4 expression or low SMARCC2 levels were associated with poor survival of NB patients. CONCLUSIONS: These findings suggest that CNBP phase separation is a target for inhibiting ribosome biogenesis and tumour progression in NB via modulating SWI/SNF complex activity.

Quality control of protein synthesis in the early elongation stage.

In the early stage of bacterial translation, peptidyl-tRNAs frequently dissociate from the ribosome (pep-tRNA drop-off) and are recycled by peptidyl-tRNA hydrolase. Here, we establish a highly sensitive method for profiling of pep-tRNAs using mass spectrometry, and successfully detect a large number of nascent peptides from pep-tRNAs accumulated in Escherichia coli pthts strain. Based on molecular mass analysis, we found about 20% of the peptides bear single amino-acid substitutions of the N-terminal sequences of E. coli ORFs. Detailed analysis of individual pep-tRNAs and reporter assay revealed that most of the substitutions take place at the C-terminal drop-off site and that the miscoded pep-tRNAs rarely participate in the next round of elongation but dissociate from the ribosome. These findings suggest that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.

Streptothricin F is a bactericidal antibiotic effective against highly drug-resistant gram-negative bacteria that interacts with the 30S subunit of the 70S ribosome.

The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.

Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance of translation.

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homolog (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in nonstarved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12 stalk base, suggesting that the aminoacylation status of A-site tRNA is surveilled during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle.

Molecular basis for recognition and deubiquitination of 40S ribosomes by Otu2.

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation.

Entropic control of the free-energy landscape of an archetypal biomolecular machine.

Biomolecular machines are complex macromolecular assemblies that utilize thermal and chemical energy to perform essential, multistep, cellular processes. Despite possessing different architectures and functions, an essential feature of the mechanisms of action of all such machines is that they require dynamic rearrangements of structural components. Surprisingly, biomolecular machines generally possess only a limited set of such motions, suggesting that these dynamics must be repurposed to drive different mechanistic steps. Although ligands that interact with these machines are known to drive such repurposing, the physical and structural mechanisms through which ligands achieve this remain unknown. Using temperature-dependent, single-molecule measurements analyzed with a time-resolution-enhancing algorithm, here, we dissect the free-energy landscape of an archetypal biomolecular machine, the bacterial ribosome, to reveal how its dynamics are repurposed to drive distinct steps during ribosome-catalyzed protein synthesis. Specifically, we show that the free-energy landscape of the ribosome encompasses a network of allosterically coupled structural elements that coordinates the motions of these elements. Moreover, we reveal that ribosomal ligands which participate in disparate steps of the protein synthesis pathway repurpose this network by differentially modulating the structural flexibility of the ribosomal complex (i.e., the entropic component of the free-energy landscape). We propose that such ligand-dependent entropic control of free-energy landscapes has evolved as a general strategy through which ligands may regulate the functions of all biomolecular machines. Such entropic control is therefore an important driver in the evolution of naturally occurring biomolecular machines and a critical consideration for the design of synthetic molecular machines.

Selective ribosome profiling as a tool to study interactions of translating ribosomes in mammalian cells.

The processing, membrane targeting and folding of newly synthesized polypeptides is closely linked to their synthesis at the ribosome. A network of enzymes, chaperones and targeting factors engages ribosome-nascent chain complexes (RNCs) to support these maturation processes. Exploring the modes of action of this machinery is critical for our understanding of functional protein biogenesis. Selective ribosome profiling (SeRP) is a powerful method for interrogating co-translational interactions of maturation factors with RNCs. It provides proteome-wide information on the factor's nascent chain interactome, the timing of factor binding and release during the progress of translation of individual nascent chain species, and the mechanisms and features controlling factor engagement. SeRP is based on the combination of two ribosome profiling (RP) experiments performed on the same cell population. In one experiment the ribosome-protected mRNA footprints of all translating ribosomes of the cell are sequenced (total translatome), while the other experiment detects only the ribosome footprints of the subpopulation of ribosomes engaged by the factor of interest (selected translatome). The codon-specific ratio of ribosome footprint densities from selected over total translatome reports on the factor enrichment at specific nascent chains. In this chapter, we provide a detailed SeRP protocol for mammalian cells. The protocol includes instructions on cell growth and cell harvest, stabilization of factor-RNC interactions, nuclease digest and purification of (factor-engaged) monosomes, as well as preparation of cDNA libraries from ribosome footprint fragments and deep sequencing data analysis. Purification protocols of factor-engaged monosomes and experimental results are exemplified for the human ribosomal tunnel exit-binding factor Ebp1 and chaperone Hsp90, but the protocols are readily adaptable to other co-translationally acting mammalian factors.

Deformylation of nascent peptide chains on the ribosome.

Processing of newly synthesized polypeptides is essential for protein homeostasis and cell viability. In bacteria and eukaryotic organelles, all proteins are synthesized with formylmethionine at their N-terminus. As the nascent peptide emerges from the ribosome during translation, the formyl group is removed by peptide deformylase (PDF), an enzyme that belongs to the family of ribosome-associated protein biogenesis factors (RPBs). Because PDF is essential in bacteria but not in humans (except for the PDF homolog acting in mitochondria), the bacterial enzyme is a promising antimicrobial drug target. While much of the mechanistic work on PDF was carried out using model peptides in solution, understanding the mechanism of PDF in cells and developing effective PDF inhibitors requires experiments with its native cellular substrates, i.e., ribosome-nascent chain complexes. Here, we describe protocols to purify PDF from Escherichia coli and to test its deformylation activity on the ribosome in multiple-turnover and single-round kinetic regimes as well as in binding assays. These protocols can be used to test PDF inhibitors, to study the peptide specificity of PDF and its interplay with other RPBs, as well as to compare the activity and specificity of bacterial and mitochondrial PDFs.