Annealing synchronizes the 70S ribosome into a minimum-energy conformation.

Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70S ribosome from Escherichia coli as a model, wherein the 30S subunit undergoes a thermally driven intersubunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70S ribosome homogeneity and improves local resolution on the 30S subunit. After annealing, the 70S ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes. Manifold-based analysis further indicates that the annealed 70S ribosome takes a narrow conformational distribution and exhibits a minimum-energy state in the free-energy landscape. Our experimental results offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing shows great potential for synchronizing proteins on a single-molecule level and can be extended to study protein folding and explore conformational and energy landscapes.

The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates.

Catch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential AAA (ATPase associated with diverse cellular activities) mechanoenzyme Mdn1 drives at least two separate steps in ribosome biogenesis, using its MIDAS domain to extract the ubiquitin-like (UBL) domain-containing proteins Rsa4 and Ytm1 from ribosomal precursors. However, it must subsequently release these assembly factors to reinitiate the enzymatic cycle. The mechanism underlying the switching of the MIDAS-UBL interaction between strongly and weakly bound states is unknown. Here, we use optical tweezers to investigate the force dependence of MIDAS-UBL binding. Parallel experiments with Rsa4 and Ytm1 show that forces up to ~4 pN, matching the magnitude of force produced by AAA proteins similar to Mdn1, enhance the MIDAS domain binding lifetime up to 10-fold, and higher forces accelerate dissociation. Together, our studies indicate that Mdn1's MIDAS domain can form catch bonds with more than one UBL substrate, and provide insights into how mechanoregulation may contribute to the Mdn1 enzymatic cycle during ribosome biogenesis.

Single-Cell and Bulk RNA-Sequencing Reveal Differences in Monocyte Susceptibility to Influenza A Virus Infection Between Africans and Europeans.

There is considerable inter-individual and inter-population variability in response to viruses. The potential of monocytes to elicit type-I interferon responses has attracted attention to their role in viral infections. Here, we use single-cell RNA-sequencing to characterize the role of cellular heterogeneity in human variation of monocyte responses to influenza A virus (IAV) exposure. We show widespread inter-individual variability in the percentage of IAV-infected monocytes. Notably, individuals with high cellular susceptibility to IAV are characterized by a lower activation at basal state of an IRF/STAT-induced transcriptional network, which includes antiviral genes such as IFITM3, MX1 and OAS3. Upon IAV challenge, we find that cells escaping viral infection display increased mRNA expression of type-I interferon stimulated genes and decreased expression of ribosomal genes, relative to both infected cells and those never exposed to IAV. We also uncover a stronger resistance of CD16+ monocytes to IAV infection, together with CD16+ -specific mRNA expression of IL6 and TNF in response to IAV. Finally, using flow cytometry and bulk RNA-sequencing across 200 individuals of African and European ancestry, we observe a higher number of CD16+ monocytes and lower susceptibility to IAV infection among monocytes from individuals of African-descent. Based on these data, we hypothesize that higher basal monocyte activation, driven by environmental factors and/or weak-effect genetic variants, underlies the lower cellular susceptibility to IAV infection of individuals of African ancestry relative to those of European ancestry. Further studies are now required to investigate how such cellular differences in IAV susceptibility translate into population differences in clinical outcomes and susceptibility to severe influenza.

LIN28B alters ribosomal dynamics to promote metastasis in MYCN-driven malignancy.

High expression of LIN28B is associated with aggressive malignancy and poor survival. Here, probing MYCN-amplified neuroblastoma as a model system, we showed that LIN28B expression was associated with enhanced cell migration in vitro and invasive and metastatic behavior in murine xenografts. Sequence analysis of the polyribosome fraction of LIN28B-expressing neuroblastoma cells revealed let-7-independent enrichment of transcripts encoding components of the translational and ribosomal apparatus and depletion of transcripts of neuronal developmental programs. We further observed that LIN28B utilizes both its cold shock and zinc finger RNA binding domains to preferentially interact with MYCN-induced transcripts of the ribosomal complex, enhancing their translation. These data demonstrated that LIN28B couples the MYCN-driven transcriptional program to enhanced ribosomal translation, thereby implicating LIN28B as a posttranscriptional driver of the metastatic phenotype.

Ribosome Elongation Kinetics of Consecutively Charged Residues Are Coupled to Electrostatic Force.

The speed of protein synthesis can dramatically change when consecutively charged residues are incorporated into an elongating nascent protein by the ribosome. The molecular origins of this class of allosteric coupling remain unknown. We demonstrate, using multiscale simulations, that positively charged residues generate large forces that move the P-site amino acid away from the A-site amino acid. Negatively charged residues generate forces of similar magnitude but move the A- and P-sites closer together. These conformational changes, respectively, increase and decrease the transition state barrier height to peptide bond formation, explaining how charged residues mechanochemically alter translation speed. This mechanochemical mechanism is consistent with in vivo ribosome profiling data exhibiting proportionality between translation speed and the number of charged residues, experimental data characterizing nascent chain conformations, and a previously published cryo-EM structure of a ribosome-nascent chain complex containing consecutive lysines. These results expand the role of mechanochemistry in translation and provide a framework for interpreting experimental results on translation speed.

First-principles model of optimal translation factors stoichiometry.

Enzymatic pathways have evolved uniquely preferred protein expression stoichiometry in living cells, but our ability to predict the optimal abundances from basic properties remains underdeveloped. Here, we report a biophysical, first-principles model of growth optimization for core mRNA translation, a multi-enzyme system that involves proteins with a broadly conserved stoichiometry spanning two orders of magnitude. We show that predictions from maximization of ribosome usage in a parsimonious flux model constrained by proteome allocation agree with the conserved ratios of translation factors. The analytical solutions, without free parameters, provide an interpretable framework for the observed hierarchy of expression levels based on simple biophysical properties, such as diffusion constants and protein sizes. Our results provide an intuitive and quantitative understanding for the construction of a central process of life, as well as a path toward rational design of pathway-specific enzyme expression stoichiometry.

Ribosome-Induced Cellular Multipotency, an Emerging Avenue in Cell Fate Reversal.

The ribosome, which is present in all three domains of life, plays a well-established, critical role in the translation process by decoding messenger RNA into protein. Ribosomal proteins, in contrast, appear to play non-translational roles in growth, differentiation, and disease. We recently discovered that ribosomes are involved in reverting cellular potency to a multipotent state. Ribosomal incorporation (the uptake of free ribosome by living cells) can direct the fate of both somatic and cancer cells into multipotency, allowing them to switch cell lineage. During this process, both types of cells experienced cell-cycle arrest and cellular stress while remaining multipotent. This review provides a molecular perspective on current insights into ribosome-induced multipotency and sheds light on how a common stress-associated mechanism may be involved. We also discuss the impact of this phenomenon on cancer cell reprogramming and its potential in cancer therapy.

Alexander Spirin on Molecular Machines and Origin of Life.

Once it was believed that ribosomal RNA encodes proteins, and GTP hydrolysis supplies the energy for protein synthesis. Everything has changed, when Alexander Spirin joined the science. It turned out that proteins are encoded by a completely different RNA, and GTP hydrolysis only accelerates the process already provided with energy. It was Spirin who first put forward the idea of a Brownian ratchet and explained how and why molecular machines could arise in the RNA world.

Ribosome as a Translocase and Helicase.

During protein synthesis, ribosome moves along mRNA to decode one codon after the other. Ribosome translocation is induced by a universally conserved protein, elongation factor G (EF-G) in bacteria and elongation factor 2 (EF-2) in eukaryotes. EF-G-induced translocation results in unwinding of the intramolecular secondary structures of mRNA by three base pairs at a time that renders the translating ribosome a processive helicase. Professor Alexander Sergeevich Spirin has made numerous seminal contributions to understanding the molecular mechanism of translocation. Here, we review Spirin's insights into the ribosomal translocation and recent advances in the field that stemmed from Spirin's pioneering work. We also discuss key remaining challenges in studies of translocase and helicase activities of the ribosome.

Structural dynamics of single SARS-CoV-2 pseudoknot molecules reveal topologically distinct conformers.

The RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target. To understand how it responds to mechanical tension applied by ribosomes, thought to play a key role during frameshifting, we probe its structural dynamics using optical tweezers. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. The pseudoknotted conformers have distinct topologies, one threading the 5' end through a 3-helix junction to create a knot-like fold, the other with unthreaded 5' end, consistent with structures observed via cryo-EM and simulations. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2; Mg2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer. These results resolve the SARS-CoV-2 frameshift signal folding mechanism and highlight its conformational heterogeneity, with important implications for structure-based drug-discovery efforts.

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.

A unifying autocatalytic network-based framework for bacterial growth laws.

Recently discovered simple quantitative relations, known as bacterial growth laws, hint at the existence of simple underlying principles at the heart of bacterial growth. In this work, we provide a unifying picture of how these known relations, as well as relations that we derive, stem from a universal autocatalytic network common to all bacteria, facilitating balanced exponential growth of individual cells. We show that the core of the cellular autocatalytic network is the transcription-translation machinery-in itself an autocatalytic network comprising several coupled autocatalytic cycles, including the ribosome, RNA polymerase, and transfer RNA (tRNA) charging cycles. We derive two types of growth laws per autocatalytic cycle, one relating growth rate to the relative fraction of the catalyst and its catalysis rate and the other relating growth rate to all the time scales in the cycle. The structure of the autocatalytic network generates numerous regimes in state space, determined by the limiting components, while the number of growth laws can be much smaller. We also derive a growth law that accounts for the RNA polymerase autocatalytic cycle, which we use to explain how growth rate depends on the inducible expression of the rpoB and rpoC genes, which code for the RpoB and C protein subunits of RNA polymerase, and how the concentration of rifampicin, which targets RNA polymerase, affects growth rate without changing the RNA-to-protein ratio. We derive growth laws for tRNA synthesis and charging and predict how growth rate depends on temperature, perturbation to ribosome assembly, and membrane synthesis.

Mitochondrial Proteostasis Requires Genes Encoded in a Neurodevelopmental Syndrome Locus.

Eukaryotic cells maintain proteostasis through mechanisms that require cytoplasmic and mitochondrial translation. Genetic defects affecting cytoplasmic translation perturb synapse development, neurotransmission, and are causative of neurodevelopmental disorders, such as Fragile X syndrome. In contrast, there is little indication that mitochondrial proteostasis, either in the form of mitochondrial protein translation and/or degradation, is required for synapse development and function. Here we focus on two genes deleted in a recurrent copy number variation causing neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. We demonstrate that SLC25A1 and MRPL40, two genes present in the microdeleted segment and whose products localize to mitochondria, interact and are necessary for mitochondrial ribosomal integrity and proteostasis. Our Drosophila studies show that mitochondrial ribosome function is necessary for synapse neurodevelopment, function, and behavior. We propose that mitochondrial proteostasis perturbations, either by genetic or environmental factors, are a pathogenic mechanism for neurodevelopmental disorders.SIGNIFICANCE STATEMENT The balance between cytoplasmic protein synthesis and degradation, or cytoplasmic proteostasis, is required for normal synapse function and neurodevelopment. Cytoplasmic and mitochondrial ribosomes are necessary for two compartmentalized, yet interdependent, forms of proteostasis. Proteostasis dependent on cytoplasmic ribosomes is a well-established target of genetic defects that cause neurodevelopmental disorders, such as autism. Here we show that the mitochondrial ribosome is a neurodevelopmentally regulated organelle whose function is required for synapse development and function. We propose that defective mitochondrial proteostasis is a mechanism with the potential to contribute to neurodevelopmental disease.

Bcl-2 Associated Athanogene 2 (BAG2) is Associated With Progression and Prognosis of Hepatocellular Carcinoma: A Bioinformatics-Based Analysis.

Background: Bcl-2 associated athanogene2 (BAG2) is reported to act as an oncogene or a tumor-suppressor in tumors in a context-dependent way; however, its function in hepatocellular carcinoma (HCC) remains unclear. Methods: Immunohistochemistry (IHC) staining, cell counting kit-8 (CCK-8) assay, apoptotic assay, cell invasion assay and a set of bioinformatics tools were integrated to analyze the role of BAG2 in hepatocellular carcinoma. Results: BAG2 was significantly up-regulated in HCC. Prognostic analysis indicated that HCC patients with high expression of BAG2 had significantly shorter overall survival, progression free survival and disease specific survival. Besides, silencing BAG2 in HCC cells impaired cell proliferation, facilitated apoptosis and repressed invasion of the cells. Bioinformatics analysis showed that BAG2 might regulate ribosome biogenesis in HCC. Conclusion: This study revealed that the up-regulated BAG2 in HCC was associated with a worse prognosis and might favor the progression of the disease.

The ribosome epitranscriptome: inert-or a platform for functional plasticity?

A universal property of all rRNAs explored to date is the prevalence of post-transcriptional ("epitranscriptional") modifications, which expand the chemical and topological properties of the four standard nucleosides. Are these modifications an inert, constitutive part of the ribosome? Or could they, in part, also regulate the structure or function of the ribosome? In this review, we summarize emerging evidence that rRNA modifications are more heterogeneous than previously thought, and that they can also vary from one condition to another, such as in the context of a cellular response or a developmental trajectory. We discuss the implications of these results and key open questions on the path toward connecting such heterogeneity with function.

How cytosolic compartments play safeguard functions against neuroinflammation and cell death in cerebral ischemia.

Ischemic stroke is the second leading cause of mortality and disability globally. Neuronal damage following ischemic stroke is rapid and irreversible, and eventually results in neuronal death. In addition to activation of cell death signaling, neuroinflammation is also considered as another pathogenesis that can occur within hours after cerebral ischemia. Under physiological conditions, subcellular organelles play a substantial role in neuronal functionality and viability. However, their functions can be remarkably perturbed under neurological disorders, particularly cerebral ischemia. Therefore, their biochemical and structural response has a determining role in the sequel of neuronal cells and the progression of disease. However, their effects on cell death and neuroinflammation, as major underlying mechanisms of ischemic stroke, are still not understood. This review aims to provide a comprehensive overview of the contribution of each organelle on these pathological processes after ischemic stroke.

Ribosome Biogenesis and Cancer: Overview on Ribosomal Proteins.

Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.

Proline codon pair selection determines ribosome pausing strength and translation efficiency in bacteria.

The speed of mRNA translation depends in part on the amino acid to be incorporated into the nascent chain. Peptide bond formation is especially slow with proline and two adjacent prolines can even cause ribosome stalling. While previous studies focused on how the amino acid context of a Pro-Pro motif determines the stalling strength, we extend this question to the mRNA level. Bioinformatics analysis of the Escherichia coli genome revealed significantly differing codon usage between single and consecutive prolines. We therefore developed a luminescence reporter to detect ribosome pausing in living cells, enabling us to dissect the roles of codon choice and tRNA selection as well as to explain the genome scale observations. Specifically, we found a strong selective pressure against CCC/U-C, a sequon causing ribosomal frameshifting even under wild-type conditions. On the other hand, translation efficiency as positive evolutionary driving force led to an overrepresentation of CCG. This codon is not only translated the fastest, but the corresponding prolyl-tRNA reaches almost saturating levels. By contrast, CCA, for which the cognate prolyl-tRNA amounts are limiting, is used to regulate pausing strength. Thus, codon selection both in discrete positions but especially in proline codon pairs can tune protein copy numbers.

Genetics animates structure: leveraging genetic interactions to study the dynamics of ribosome biogenesis.

The assembly of eukaryotic ribosomes follows an assembly line-like pathway in which numerous trans-acting biogenesis factors act on discrete pre-ribosomal intermediates to progressively shape the nascent subunits into their final functional architecture. Recent advances in cryo-electron microscopy have led to high-resolution structures of many pre-ribosomal intermediates; however, these static snapshots do not capture the dynamic transitions between these intermediates. To this end, molecular genetics can be leveraged to reveal how the biogenesis factors drive these dynamic transitions. Here, we briefly review how we recently used the deletion of BUD23 (bud23∆) to understand its role in the assembly of the ribosomal small subunit. The strong growth defect of bud23∆ mutants places a selective pressure on yeast cells for the occurrence of extragenic suppressors that define a network of functional interactions among biogenesis factors. Mapping these suppressing mutations to recently published structures of pre-ribosomal complexes allowed us to contextualize these suppressing mutations and derive a detailed model in which Bud23 promotes a critical transition event to facilitate folding of the central pseudoknot of the small subunit. This mini-review highlights how genetics can be used to understand the dynamics of complex structures, such as the maturing ribosome.

Using prototyping to choose a bioinformatics workflow management system.

Workflow management systems represent, manage, and execute multistep computational analyses and offer many benefits to bioinformaticians. They provide a common language for describing analysis workflows, contributing to reproducibility and to building libraries of reusable components. They can support both incremental build and re-entrancy-the ability to selectively re-execute parts of a workflow in the presence of additional inputs or changes in configuration and to resume execution from where a workflow previously stopped. Many workflow management systems enhance portability by supporting the use of containers, high-performance computing (HPC) systems, and clouds. Most importantly, workflow management systems allow bioinformaticians to delegate how their workflows are run to the workflow management system and its developers. This frees the bioinformaticians to focus on what these workflows should do, on their data analyses, and on their science. RiboViz is a package to extract biological insight from ribosome profiling data to help advance understanding of protein synthesis. At the heart of RiboViz is an analysis workflow, implemented in a Python script. To conform to best practices for scientific computing which recommend the use of build tools to automate workflows and to reuse code instead of rewriting it, the authors reimplemented this workflow within a workflow management system. To select a workflow management system, a rapid survey of available systems was undertaken, and candidates were shortlisted: Snakemake, cwltool, Toil, and Nextflow. Each candidate was evaluated by quickly prototyping a subset of the RiboViz workflow, and Nextflow was chosen. The selection process took 10 person-days, a small cost for the assurance that Nextflow satisfied the authors' requirements. The use of prototyping can offer a low-cost way of making a more informed selection of software to use within projects, rather than relying solely upon reviews and recommendations by others.