Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro.

Stress-induced molecular damage to ribosomes can impact protein synthesis in cells, but cell-based assays do not provide a clear way to distinguish the effects of ribosome damage from stress responses and damage to other parts of the translation machinery. Here we describe a detailed protocol for the separation of yeast ribosomes from other translational machinery constituents, followed by reconstitution of the translation mixture in vitro. This technique, which we refer to as ribosome separation and reconstitution (RSR), allows chemical modifications of yeast ribosomes without compromising other key translational components. In addition to the characterization of stress-induced ribosome damage, RSR can be applied to a broad range of experimental problems in studies of yeast translation.

Interchangeable utilization of metals: New perspectives on the impacts of metal ions employed in ancient and extant biomolecules.

Metal ions provide considerable functionality across biological systems, and their utilization within biomolecules has adapted through changes in the chemical environment to maintain the activity they facilitate. While ancient earth's atmosphere was rich in iron and manganese and low in oxygen, periods of atmospheric oxygenation significantly altered the availability of certain metal ions, resulting in ion replacement within biomolecules. This adaptation mechanism has given rise to the phenomenon of metal cofactor interchangeability, whereby contemporary proteins and nucleic acids interact with multiple metal ions interchangeably, with different coordinated metals influencing biological activity, stability, and toxic potential. The ability of extant organisms to adapt to fluctuating metal availability remains relevant in a number of crucial biomolecules, including the superoxide dismutases of the antioxidant defense systems and ribonucleotide reductases. These well-studied and ancient enzymes illustrate the potential for metal interchangeability and adaptive utilization. More recently, the ribosome has also been demonstrated to exhibit interchangeable interactions with metal ions with impacts on function, stability, and stress adaptation. Using these and other examples, here we review the biological significance of interchangeable metal ions from a new angle that combines both biochemical and evolutionary viewpoints. The geochemical pressures and chemical properties that underlie biological metal utilization are discussed in the context of their impact on modern disease states and treatments.

Proteotoxic stress promotes entrapment of ribosomes and misfolded proteins in a shared cytosolic compartment.

Cells continuously monitor protein synthesis to prevent accumulation of aberrant polypeptides. Insufficient capacity of cellular degradative systems, chaperone shortage or high levels of mistranslation by ribosomes can result in proteotoxic stress and endanger proteostasis. One of the least explored reasons for mistranslation is the incorrect functioning of the ribosome itself. To understand how cells deal with ribosome malfunction, we introduced mutations in the Expansion Segment 7 (ES7L) of 25S rRNA that allowed the formation of mature, translationally active ribosomes but induced proteotoxic stress and compromised cell viability. The ES7L-mutated ribosomes escaped nonfunctional rRNA Decay (NRD) and remained stable. Remarkably, ES7L-mutated ribosomes showed increased segregation into cytoplasmic foci containing soluble misfolded proteins. This ribosome entrapment pathway, termed TRAP (Translational Relocalization with Aberrant Polypeptides), was generalizable beyond the ES7L mutation, as wild-type ribosomes also showed increased relocalization into the same compartments in cells exposed to proteotoxic stressors. We propose that during TRAP, assembled ribosomes associated with misfolded nascent chains move into cytoplasmic compartments enriched in factors that facilitate protein quality control. In addition, TRAP may help to keep translation at its peak efficiency by preventing malfunctioning ribosomes from active duty in translation.

Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese.

Protein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg2+ for structure. Recent work has indicated that other metal cations can substitute for Mg2+, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe2+ and the ribosome and identify Mn2+ as a factor capable of attenuating oxidant-induced Fe2+-mediated degradation of rRNA. We report that Fe2+ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn2+ competes with Fe2+ for rRNA-binding sites and that protection of ribosomes from Fe2+-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress.

Cooperativity between the Ribosome-Associated Chaperone Ssb/RAC and the Ubiquitin Ligase Ltn1 in Ubiquitination of Nascent Polypeptides.

Eukaryotic cells have evolved multiple mechanisms to detect and eliminate aberrant polypeptides. Co-translational protein surveillance systems play an important role in these mechanisms. These systems include ribosome-associated protein quality control (RQC) that detects aberrant nascent chains stalled on ribosomes and promotes their ubiquitination and degradation by the proteasome, and ribosome-associated chaperone Ssb/RAC, which ensures correct nascent chain folding. Despite the known function of RQC and Ssb/ribosome-associated complex (RAC) in monitoring the quality of newly generated polypeptides, whether they cooperate during initial stages of protein synthesis remains unexplored. Here, we provide evidence that Ssb/RAC and the ubiquitin ligase Ltn1, the major component of RQC, display genetic and functional cooperativity. Overexpression of Ltn1 rescues growth suppression of the yeast strain-bearing deletions of SSB genes during proteotoxic stress. Moreover, Ssb/RAC promotes Ltn1-dependent ubiquitination of nascent chains associated with 80S ribosomal particles but not with translating ribosomes. Consistent with this finding, quantitative western blot analysis revealed lower levels of Ltn1 associated with 80S ribosomes and with free 60S ribosomal subunits in the absence of Ssb/RAC. We propose a mechanism in which Ssb/RAC facilitates recruitment of Ltn1 to ribosomes, likely by detecting aberrations in nascent chains and leading to their ubiquitination and degradation.

Effects of Oxidative Stress on Protein Translation: Implications for Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are a group of disorders that affect the heart and blood vessels. Due to their multifactorial nature and wide variation, CVDs are the leading cause of death worldwide. Understanding the molecular alterations leading to the development of heart and vessel pathologies is crucial for successfully treating and preventing CVDs. One of the causative factors of CVD etiology and progression is acute oxidative stress, a toxic condition characterized by elevated intracellular levels of reactive oxygen species (ROS). Left unabated, ROS can damage virtually any cellular component and affect essential biological processes, including protein synthesis. Defective or insufficient protein translation results in production of faulty protein products and disturbances of protein homeostasis, thus promoting pathologies. The relationships between translational dysregulation, ROS, and cardiovascular disorders will be examined in this review.

Aggregation and Prion-Inducing Properties of the G-Protein Gamma Subunit Ste18 are Regulated by Membrane Association.

Yeast prions and mnemons are respectively transmissible and non-transmissible self-perpetuating protein assemblies, frequently based on cross-ß ordered detergent-resistant aggregates (amyloids). Prions cause devastating diseases in mammals and control heritable traits in yeast. It was shown that the de novo formation of the prion form [PSI+] of yeast release factor Sup35 is facilitated by aggregates of other proteins. Here we explore the mechanism of the promotion of [PSI+] formation by Ste18, an evolutionarily conserved gamma subunit of a G-protein coupled receptor, a key player in responses to extracellular stimuli. Ste18 forms detergent-resistant aggregates, some of which are colocalized with de novo generated Sup35 aggregates. Membrane association of Ste18 is required for both Ste18 aggregation and [PSI+] induction, while functional interactions involved in signal transduction are not essential for these processes. This emphasizes the significance of a specific location for the nucleation of protein aggregation. In contrast to typical prions, Ste18 aggregates do not show a pattern of heritability. Our finding that Ste18 levels are regulated by the ubiquitin-proteasome system, in conjunction with the previously reported increase in Ste18 levels upon the exposure to mating pheromone, suggests that the concentration-dependent Ste18 aggregation may mediate a mnemon-like response to physiological stimuli.

Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.

Stress-induced strand breaks in rRNA have been observed in many organisms, but the mechanisms by which they originate are not well-understood. Here we show that a chemical rather than an enzymatic mechanism initiates rRNA cleavages during oxidative stress in yeast (Saccharomyces cerevisiae). We used cells lacking the mitochondrial glutaredoxin Grx5 to demonstrate that oxidant-induced cleavage formation in 25S rRNA correlates with intracellular iron levels. Sequestering free iron by chemical or genetic means decreased the extent of rRNA degradation and relieved the hypersensitivity of grx5Δ cells to the oxidants. Importantly, subjecting purified ribosomes to an in vitro iron/ascorbate reaction precisely recapitulated the 25S rRNA cleavage pattern observed in cells, indicating that redox activity of the ribosome-bound iron is responsible for the strand breaks in the rRNA. In summary, our findings provide evidence that oxidative stress-associated rRNA cleavages can occur through rRNA strand scission by redox-active, ribosome-bound iron that potentially promotes Fenton reaction-induced hydroxyl radical production, implicating intracellular iron as a key determinant of the effects of oxidative stress on ribosomes. We propose that iron binding to specific ribosome elements primes rRNA for cleavages that may play a role in redox-sensitive tuning of the ribosome function in stressed cells.

Endonucleolytic cleavage in the expansion segment 7 of 25S rRNA is an early marker of low-level oxidative stress in yeast.

The ability to detect and respond to oxidative stress is crucial to the survival of living organisms. In cells, sensing of increased levels of reactive oxygen species (ROS) activates many defensive mechanisms that limit or repair damage to cell components. The ROS-signaling responses necessary for cell survival under oxidative stress conditions remain incompletely understood, especially for the translational machinery. Here, we found that drug treatments or a genetic deficiency in the thioredoxin system that increase levels of endogenous hydrogen peroxide in the yeast Saccharomyces cerevisiae promote site-specific endonucleolytic cleavage in 25S ribosomal RNA (rRNA) adjacent to the c loop of the expansion segment 7 (ES7), a putative regulatory region located on the surface of the 60S ribosomal subunit. Our data also show that ES7c is cleaved at early stages of the gene expression program that enables cells to successfully counteract oxidative stress and is not a prerequisite or consequence of apoptosis. Moreover, the 60S subunits containing ES7c-cleaved rRNA cofractionate with intact subunits in sucrose gradients and repopulate polysomes after a short starvation-induced translational block, indicating their active role in translation. These results demonstrate that ES7c cleavage in rRNA is an early and sensitive marker of increased ROS levels in yeast cells and suggest that changes in ribosomes may be involved in the adaptive response to oxidative stress.

Distinct types of translation termination generate substrates for ribosome-associated quality control.

Cotranslational degradation of polypeptide nascent chains plays a critical role in quality control of protein synthesis and the rescue of stalled ribosomes. In eukaryotes, ribosome stalling triggers release of 60S subunits with attached nascent polypeptides, which undergo ubiquitination by the E3 ligase Ltn1 and proteasomal degradation facilitated by the ATPase Cdc48. However, the identity of factors acting upstream in this process is less clear. Here, we examined how the canonical release factors Sup45-Sup35 (eRF1-eRF3) and their paralogs Dom34-Hbs1 affect the total population of ubiquitinated nascent chains associated with yeast ribosomes. We found that the availability of the functional release factor complex Sup45-Sup35 strongly influences the amount of ubiquitinated polypeptides associated with 60S ribosomal subunits, while Dom34-Hbs1 generate 60S-associated peptidyl-tRNAs that constitute a relatively minor fraction of Ltn1 substrates. These results uncover two separate pathways that target nascent polypeptides for Ltn1-Cdc48-mediated degradation and suggest that in addition to canonical termination on stop codons, eukaryotic release factors contribute to cotranslational protein quality control.

Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA.

Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5'-truncated 5.8S rRNA, which we named 5.8SC. The 5' exoribonuclease Xrn2 is involved in the generation of both 5.8S(C) and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8S(C) rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes.

One-step hot formamide extraction of RNA from Saccharomyces cerevisiae.

Current methods for isolating RNA from budding yeast require lengthy and laborious steps such as freezing and heating with phenol, homogenization with glass beads, or enzymatic digestion of the cell wall. Here, extraction with a solution of formamide and EDTA was adapted to isolate RNA from whole yeast cells through a rapid and easily scalable procedure that does not require mechanical cell lysis, phenol, or enzymes. RNA extracted with formamide-EDTA can be directly loaded on gels for electrophoretic analysis without alcohol precipitation. A simplified protocol for downstream DNase treatment and reverse transcription reaction is also included. The formamide-EDTA extraction of yeast RNA is faster, safer, and more economical than conventional methods, outperforms them in terms of total yield, and greatly increases throughput.

Golgi-mediated glycosylation determines residency of the T2 RNase Rny1p in Saccharomyces cerevisiae.

The role of glycosylation in the function of the T2 family of RNases is not well understood. In this work, we examined how glycosylation affects the progression of the T2 RNase Rny1p through the secretory pathway in Saccharomyces cerevisiae. We found that Rny1p requires entering into the ER first to become active and uses the adaptor protein Erv29p for packaging into COPII vesicles and transport to the Golgi apparatus. While inside the ER, Rny1p undergoes initial N-linked core glycosylation at four sites, N37, N70, N103 and N123. Rny1p transport to the Golgi results in the further attachment of high-glycans. Whereas modifications with glycans are dispensable for the nucleolytic activity of Rny1p, Golgi-mediated modifications are critical for its extracellular secretion. Failure of Golgi-specific glycosylation appears to direct Rny1p to the vacuole as an alternative destination and/or site of terminal degradation. These data reveal a previously unknown function of Golgi glycosylation in a T2 RNase as a sorting and secretion signal.

The ubiquitin ligase Rsp5 is required for ribosome stability in Saccharomyces cerevisiae.

Rsp5p is a conserved HECT-domain ubiquitin ligase with diverse roles in cellular physiology. Here we report a previously unknown role of Rsp5p in facilitating the stability of the cytoplasmic ribosome pool in budding yeast. Yeast strains carrying temperature-sensitive mutations in RSP5 showed a progressive decline in levels of 18S and 25S rRNAs and accumulation of rRNA decay fragments when cells grown in rich medium were shifted to restrictive temperature. This was accompanied by a decreased number of translating ribosomes and the appearance of ribosomal subunits with an abnormally low sedimentation rate in polysome analysis. Abrogating Rsp5p function affected stability of other tested noncoding RNA species (tRNA and snoRNA), but to a lower extent than that of rRNA, and also inhibited processing of rRNA and tRNA precursors, in agreement with previous studies. The breakdown of cellular ribosomes was not affected by deletion of key genes involved in autophagy, previously implicated in ribosome turnover upon starvation. Our results suggest that functional Rsp5p is required to maintain the integrity of cytoplasmic ribosomes under rich nutrient conditions.

HIV-1 gp120 protein promotes HAND through the calcineurin pathway activation.

For over two decades, highly active antiretroviral therapy (HAART) was able to help prolong the life expectancy of people living with HIV-1 (PLWH) and eliminate the virus to an undetectable level. However, an increased prevalence of HIV- associated neurocognitive disorders (HAND) was observed. These symptoms range from neuronal dysfunction to cell death. Among the markers of neuronal deregulation, we cite the alteration of synaptic plasticity and neuronal communications. Clinically, these dysfunctions led to neurocognitive disorders such as learning alteration and loss of spatial memory, which promote premature brain aging even in HAART-treated patients. In support of these observations, we showed that the gp120 protein deregulates miR-499-5p and its downstream target, the calcineurin (CaN) protein. The gp120 protein also promotes the accumulation of calcium (Ca2+) and reactive oxygen species (ROS) inside the neurons leading to the activation of CaN and the inhibition of miR-499-5p. gp120 protein also caused mitochondrial fragmentation and changes in shape and size. The use of mimic miR-499 restored mitochondrial functions, appearance, and size. These results demonstrated the additional effect of the gp120 protein on neurons through the miR-499-5p/calcineurin pathway.

Profiling and verifying the substrates of E3 ubiquitin ligase Rsp5 in yeast cells.

Yeast is an essential model organism for studying protein ubiquitination pathways; however, identifying the direct substrates of E3 in the cell presents a challenge. Here, we present a protocol for using the orthogonal ubiquitin transfer (OUT) cascade to profile the substrate specificity of yeast E3 Rsp5. We describe steps for OUT profiling, proteomics analysis, in vitro and in cell ubiquitination, and stability assay. The protocol can be adapted for identifying and verifying the ubiquitination targets of other E3s in yeast. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells.

Most transcripts in growing cells are ribosomal RNA precursors (pre-rRNA). Here, we show that in mammals, aberrant pre-rRNA transcripts generated by RNA polymerase I (Pol I) are polyadenylated and accumulate markedly after treatment with low concentrations of actinomycin D (ActD), which blocks the synthesis of full-length rRNA. The poly(A) polymerase-associated domain-containing protein 5 is required for polyadenylation, whereas the exosome is partly responsible for the degradation of the short aberrant transcripts. Thus, polyadenylation functions in the quality control of Pol I transcription in metazoan cells. The impact of excessive aberrant RNAs on the degradation machinery is an unrecognized mechanism that might contribute to biological properties of ActD.

SARS-CoV-2 Causes Lung Inflammation through Metabolic Reprogramming and RAGE.

Clinical studies indicate that patients infected with SARS-CoV-2 develop hyperinflammation, which correlates with increased mortality. The SARS-CoV-2/COVID-19-dependent inflammation is thought to occur via increased cytokine production and hyperactivity of RAGE in several cell types, a phenomenon observed for other disorders and diseases. Metabolic reprogramming has been shown to contribute to inflammation and is considered a hallmark of cancer, neurodegenerative diseases, and viral infections. Malfunctioning glycolysis, which normally aims to convert glucose into pyruvate, leads to the accumulation of advanced glycation end products (AGEs). Being aberrantly generated, AGEs then bind to their receptor, RAGE, and activate several pro-inflammatory genes, such as IL-1b and IL-6, thus, increasing hypoxia and inducing senescence. Using the lung epithelial cell (BEAS-2B) line, we demonstrated that SARS-CoV-2 proteins reprogram the cellular metabolism and increase pyruvate kinase muscle isoform 2 (PKM2). This deregulation promotes the accumulation of AGEs and senescence induction. We showed the ability of the PKM2 stabilizer, Tepp-46, to reverse the observed glycolysis changes/alterations and restore this essential metabolic process.

Short and Sweet: Viral 5`-UTR as a Canonical and Non-Canonical Translation Initiation Switch.

The replication of viruses requires host cell functions, specifically for protein synthesis, as viruses lack their own translational machinery. Failure to translate viral mRNAs and generate viral proteins would affect the propagation and evolution of a virus. Thus, independently of their size, complexity, and genomes, viruses evolved sophisticated molecular mechanisms to hijack the translational apparatus of a host in order to recruit ribosomes for efficient protein production. One of the prevalent mechanisms of translation regulation utilized by viruses is non-canonical translation initiation. It is often governed by the 5'-untranslated regions (5'-UTRs) present upstream of a protein-coding sequence in viral mRNAs, such as internal ribosome entry sites (IRESs) and capindependent translation enhancers (CITEs). Viruses can also utilize canonical translation initiation factors of a host in non-canonical ways. Understanding strategies and mechanisms used by viruses to generate proteins is an important task, as it might help develop new therapeutic interventions. We previously have demonstrated that mRNA from the genome of the black beetle virus (BBV) of the Nodaviridae family contains short and unstructured 5'-UTR, which governs translation initiation as a CITE and as a canonical translational enhancer. In this Commentary, we summarize cap-dependent and cap-independent translation initiation mechanisms and further elaborate on the unique ability of the BBV mRNA 5'-UTR to switch between these two modes of translation initiation in the context of the viral life cycle. Medical implications in treating the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection by targeting viral 5'-UTRs are also discussed.

Cell-free Translation: Preparation and Validation of Translation-competent Extracts from Saccharomyces cerevisiae.

Cell-free translation is a powerful technique for in vitro protein synthesis. While cell-free translation platforms prepared from bacterial, plant, and mammalian cells are commercially available, yeast-based translation systems remain proprietary knowledge of individual labs. Here, we provide a detailed protocol for simple, fast, and cost-effective preparation of the translation-competent cell-free extract (CFE) from budding yeast. Our protocol streamlines steps combined from different procedures published over the last three decades and incorporates cryogenic lysis of yeast cells to produce a high yield of the translationally active material. We also describe techniques for the validation and troubleshooting of the quality and translational activity of the obtained yeast CFE. Graphic abstract: The flow of Cell-Free Extract (CFE) preparation procedure.