70S Ribosome Research Articles

MTR4 adaptor PICT1 functions in two distinct steps during pre-rRNA processing

Ribosome biogenesis proceeds with the successive cleavage and trimming of the large 47S rRNA precursor, where the RNA exosome plays major roles in concert with the Ski2-like RNA helicase, MTR4. The recent finding of a consensus amino acid sequence, the arch-interacting motif (AIM), for binding to the arch domain in MTR4 suggests that recruitment of the RNA processing machinery to the maturing pre-rRNA at appropriate places and timings is mediated by several adaptor proteins possessing the AIM sequence. In yeast Saccharomyces cerevisiae, Nop53 plays such a role in the maturation of the 3′-end of 5.8S rRNA. Here, we investigated the functions of PICT1 (also known as GLTSCR2 or NOP53), a mammalian ortholog of Nop53, during ribosome biogenesis in human cells. PICT1 interacted with MTR4 and exosome in an AIM-dependent manner. Overexpression of PICT1 mutants defecting AIM sequence and siRNA-mediated depletion of PICT1 showed that PICT1 is involved in two distinct pre-rRNA processing steps during the generation of 60S ribosomes; first step is the early cleavage of 32S intermediate RNA, while the second step is the late maturation of 12S precursor into 5.8S rRNA. The recruitment of MTR4 and RNA exosome via the AIM sequence was required only during the late processing step. Although, the depletion of MTR4 and PICT1 induced stabilization of the tumor suppressor p53 protein in cancer cell lines, the depletion of the exosome catalytic subunits, RRP6 and DIS3, did not exert such an effect. These results suggest that recruitment of the RNA processing machinery to the 3′-end of pre-5.8S rRNA may be involved in the induction of the nucleolar stress response, but the pre-rRNA processing capabilities themselves were not involved in this process.

Single-molecule dynamics suggest that ribosomes assemble at sites of translation in Bacillus subtilis.

Eukaryotic cells transcribe ribosomal RNA and largely assemble ribosomes in a structure called the nucleolus, where chromosomal regions containing rRNA operons are clustered. In bacteria, many rRNA operons cluster close to the origin regions that are positioned on the outer borders of nucleoids, close to polar areas, where translating 70S ribosomes are located. Because outer regions of the nucleoids contain the highest accumulation of RNA polymerase, it has been hypothesized that bacteria contain "nucleolus-like" structures. However, ribosome subunits freely diffuse through the entire cells, and could thus be assembled and matured throughout the non-compartmentalized cell. By tracking single molecules of two GTPases that play an essential role in ribosomal folding and processing in Bacillus subtilis, we show that this process takes place at sites of translation, i.e., predominantly at the cell poles. Induction of the stringent response led to a change in the population of GTPases assumed to be active in maturation, but did not abolish nucleoid occlusion of ribosomes or of GTPases. Our findings strongly support the idea of the conceptualization of nucleolus-like structures in bacteria, i.e., rRNA synthesis, ribosomal protein synthesis and subunit assembly occurring in close proximity at the cell poles, facilitating the efficiency of ribosome maturation even under conditions of transient nutrient deprivation.

Amplification of Amyloid Protein-induced Aggregation of the Eukaryotic Ribosome.

Alzheimer's disease (AD) is characterized by the aggregation of Tau protein and Amyloid-β peptides (Aβ 1-40 and Aβ 1-42). A loss of ribosomal population is also observed in the neurons in affected regions of AD. Our studies demonstrated that in vitro aggregation of amyloid forming proteins, Aβ peptides and Tau protein variants (AFPs), in the vicinity of yeast 80S ribosome can induce co-aggregation of ribosomal components. In this study, the ability of minute quantities of AFP-ribosome co-aggregates to seed the aggregation of a large excess of untreated 80S ribosomes was explored. The AFPs were purified using ion-exchange chromatography. Seeded aggregation of ribosomes in the presence of minute quantities of ribosome-protein co-aggregates or ribosomal components was studied using agarose gel electrophoretic and SDS-PAGE analysis of the pellets and Sucrose Density Gradient centrifugation of the supernatant obtained after centrifugation of the aggregation reaction mixture. Our studies, therefore, demonstrate that minute quantities of AFP-80S co-aggregate have significant seeding potential and could lead to aggregation of a large excess of fresh 80S ribosomes and this seeding ability is sustained over multiple cycles of ribosome aggregation. The aggregation propensity of ribosomal components alone could contribute towards the seeding of ribosome aggregation. The ability of minute quantities of AFP-80S co-aggregates to seed the aggregation of a large excess of fresh 80S ribosomes would result in the loss of global ribosomal population in Alzheimer's disease afflicted neurons. Hence, subject to further validation by in vivo studies, our in vitro studies indicate a significant mode of toxicity of amyloid aggregates that might be important in Alzheimer's disease pathology.

Mycn regulates intestinal development through ribosomal biogenesis in a zebrafish model of Feingold syndrome 1.

Feingold syndrome type 1, caused by loss-of-function of MYCN, is characterized by varied phenotypes including esophageal and duodenal atresia. However, no adequate model exists for studying the syndrome's pathological or molecular mechanisms, nor is there a treatment strategy. Here, we developed a zebrafish Feingold syndrome type 1 model with nonfunctional mycn, which had severe intestinal atresia. Single-cell RNA-seq identified a subcluster of intestinal cells that were highly sensitive to Mycn, and impaired cell proliferation decreased the overall number of intestinal cells in the mycn mutant fish. Bulk RNA-seq and metabolomic analysis showed that expression of ribosomal genes was down-regulated and that amino acid metabolism was abnormal. Northern blot and ribosomal profiling analysis showed abnormal rRNA processing and decreases in free 40S, 60S, and 80S ribosome particles, which led to impaired translation in the mutant. Besides, both Ribo-seq and western blot analysis showed that mTOR pathway was impaired in mycn mutant, and blocking mTOR pathway by rapamycin treatment can mimic the intestinal defect, and both L-leucine and Rheb, which can elevate translation via activating TOR pathway, could rescue the intestinal phenotype of mycn mutant. In summary, by this zebrafish Feingold syndrome type 1 model, we found that disturbance of ribosomal biogenesis and blockage of protein synthesis during development are primary causes of the intestinal defect in Feingold syndrome type 1. Importantly, our work suggests that leucine supplementation may be a feasible and easy treatment option for this disease.

Structural Consequences of Deproteinating the 50S Ribosome

Ribosomes are complex ribonucleoprotein particles. Purified 50S ribosomes subjected to high-salt wash, removing a subset of ribosomal proteins (r-proteins), were shown as competent for in vitro assembly into functional 50S subunits. Here, we used cryo-EM to determine the structures of such LiCl core particles derived from E. coli 50S subunits. A wide range of complexes with large variations in the extent of the ordered 23S rRNA and the occupancy of r-proteins were resolved to between 2.8 Å and 9 Å resolution. Many of these particles showed high similarity to in vivo and in vitro assembly intermediates, supporting the inherent stability or metastability of these states. Similar to states in early ribosome assembly, the main class showed an ordered density for the particle base around the exit tunnel, with domain V and the 3'-half of domain IV disordered. In addition, smaller core particles were discovered, where either domain II or IV was unfolded. Our data support a multi-pathway in vitro disassembly process, similar but reverse to assembly. Dependencies between complex tertiary RNA structures and RNA-protein interactions were observed, where protein extensions dissociated before the globular domains. We observed the formation of a non-native RNA structure upon protein dissociation, demonstrating that r-proteins stabilize native RNA structures and prevent non-native interactions also after folding.

A noncanonical RNA-binding domain of the fragile X protein, FMRP, elicits translational repression independent of mRNA G-quadruplexes

Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome, the leading form of inherited intellectual disability and the most common monogenic cause of autism spectrum disorders. FMRP is an RNA-binding protein that controls neuronal mRNA localization and translation. FMRP is thought to inhibit translation elongation after being recruited to target transcripts via binding RNA G-quadruplexes (G4s) within the coding sequence. Here, we directly test this model and report that FMRP inhibits translation independent of mRNA G4s. Furthermore, we found that the RGG box motif together with its natural C-terminal domain forms a noncanonical RNA-binding domain (ncRBD) that is essential for translational repression. The ncRBD elicits broad RNA-binding ability and binds to multiple reporter mRNAs and all four homopolymeric RNAs. Serial deletion analysis of the ncRBD identified that the regions required for mRNA binding and translational repression overlap but are not identical. Consistent with FMRP stalling elongating ribosomes and causing the accumulation of slowed 80S ribosomes, transcripts bound by FMRP via the ncRBD cosediment with heavier polysomes and were present in puromycin-resistant ribosome complexes. Together, this work identifies a ncRBD and translational repression domain that shifts our understanding of how FMRP inhibits translation independent of mRNA G4s.

Structural basis for HflXr-mediated antibiotic resistance in Listeria monocytogenes.

HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than inHflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.

Recent Advancements in Bottromycin Biosynthesis

AbstractBottromycin is a structurally complex cyclic peptidic compound isolated from Streptomyces bottropensis and related organisms and belongs to the RiPP family of natural products (ribosomally synthesized and post-translationally modified peptides). It exhibits potent antibacterial properties against gram-positive pathogens (including drug resistant strains such as MRSA, MIC 1 μg/mL and VRE, MIC 0.5 μg/mL) and mycoplasma. Bottromycin blocks the binding of the aminoacyl-tRNA to the A-site on the 50S ribosome and hence inhibits protein synthesis. Bottromycins contain structurally diverse post-translational modifications (PTMs) on a small peptide (GPVVVFDC) including a unique macrocyclic amidine, rare β-methylation, terminal thiazole heterocycle, oxidative decarboxylation, and Asp epimerization, among others. It exhibits a precursor peptide organization with a C-terminal follower peptide and a N-terminal core peptide. There are several new studies reported recently which gave detailed insights into the bottromycin biosynthesis pathway. This Account highlights the current advancements in understanding the biosynthetic pathway of bottromycin focusing mainly on the biochemically and structurally characterized enzymes and intricate details of the peptide–protein biophysical interactions. These studies have provided a strong foundation for conducting combinatorial biosynthesis and synthetic biological studies to create novel bottromycin variants for therapeutic applications.1 Introduction2 Biosynthetic Pathway for Bottromycin3 Enzymology of Bottromycin Biosynthesis3.1 Cleavage of Methionine (BotP)3.2 Radical SAM Methyltransferases (BotRMT1, BotRMT2, BotRMT3)3.3 ATP-Dependent YcaO Enzymes3.3.1 Thiazoline Formation by BotC3.3.2 Macrolactamidine Formation by BotCD3.4 Follower Peptide Hydrolysis (BotAH)3.5 Aspartate Epimerization (BotH)3.6 Oxidative Decarboxylation (BotCYP)3.7 O-Methyltransferase (BotOMT)4 Heterologous Bottromycin Production and Analogue Preparation5 Summary and Outlook

Bidirectional sequestration between a bacterial hibernation factor and a glutamate metabolizing protein

Bacterial hibernating 100S ribosomes (the 70S dimers) are excluded from translation and are protected from ribonucleolytic degradation, thereby promoting long-term viability and increased regrowth. No extraribosomal target of any hibernation factor has been reported. Here, we discovered a previously unrecognized binding partner (YwlG) of hibernation-promoting factor (HPF) in the human pathogen Staphylococcus aureus. YwlG is an uncharacterized virulence factor in S. aureus. We show that the HPF-YwlG interaction is direct, independent of ribosome binding, and functionally linked to cold adaptation and glucose metabolism. Consistent with the distant resemblance of YwlG to the hexameric structures of nicotinamide adenine dinucleotide (NAD)-specific glutamate dehydrogenases (GDHs), YwlG overexpression can compensate for a loss of cellular GDH activity. The reduced abundance of 100S complexes and the suppression of YwlG-dependent GDH activity provide evidence for a two-way sequestration between YwlG and HPF. These findings reveal an unexpected layer of regulation linking the biogenesis of 100S ribosomes to glutamate metabolism.

Backbone and side chain NMR assignments for the ribosome maturation factor P (RimP) from Staphylococcus aureus.

The ribosomal maturation factor (RimP) is a 17.7kDa protein and is the assembly factor of the 30S subunit. RimP is essential for efficient processing of 16S rRNA and maturation (assembly) of the 30S ribosome. It was suggested that RimP takes part in stabilization of the central pseudoknot at the early stages of the 30S subunit maturation, and this process may occur before the head domain assembly and later stages of the 30S assembly, but the mechanism of this interaction is still not fully understood. Here we report the assignment of the 1H, 13C and 15N chemical shift in the backbone and side chains of RimP from Staphylococcus aureus. Analysis of chemical shifts of the main chain using TALOS + suggests that the RimP contains eight β-strands and three α-helices with the topology α1-β1-β2-α2- β3- α3- β4- β5- β6- β7- β8. Structural studies of RimP and its complex with the ribosome by integrated structural biology approaches (NMR spectroscopy, X-ray diffraction analysis and cryoelectron microscopy) will allow further screening of highly selective inhibitors of the translation of S. aureus.

Conjugates of Desmycosin with Fragments of Antimicrobial Peptide Oncocin: Synthesis, Antibacterial Activity, Interaction with Ribosome.

Design and synthesis of conjugates consisting of the macrolide antibiotic desmycosin and fragments of the antibacterial peptide oncocin were performed in attempt to develop new antimicrobial compounds. New compounds were shown to bind to the E. coli 70S ribosomes, to inhibit bacterial protein synthesis in vitro, as well as to suppress bacterial growth. The conjugates of N-terminal hexa- and tripeptide fragments of oncocin and 3,2',4''-triacetyldesmycosin were found to be active against some strains of macrolide-resistant bacteria. By simulating molecular dynamics of the complexes of these compounds with the wild-type bacterial ribosomes and with ribosomes, containing A2059G 23S RNA mutation, the specific structural features of their interactions were revealed.

Sensing of individual stalled 80S ribosomes by Fap1 for nonfunctional rRNA turnover

Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on noncolliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S nonfunctional rRNA decay via polyubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also an association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting an mRNA stasis sensing activity, and Fap1 sterically hinders the formationof canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.

Neuronal RNA granules are ribosome complexes stalled at the pre-translocation state

The polarized cell morphology of neurons dictates many neuronal processes, including the axodendridic transport of specific mRNAs and subsequent translation. mRNAs together with ribosomes and RNA-binding proteins form RNA granules that are targeted to axodendrites for localized translation in neurons. It has been established that localized protein synthesis in neurons is essential for long-term memory formation, synaptic plasticity, and neurodegeneration. We have used proteomics and electron microscopy to characterize neuronal RNA granules (nRNAg) isolated from rat brain tissues or human neuroblastoma. We show that ribosome-containing RNA granules are morula-like structures when visualized by electron microscopy. Crosslinking-coupled mass-spectrometry identified a potential G3BP2 binding site on the ribosome near the eIF3d-binding site on the 40S ribosomal subunit. We used cryo-EM to resolve the structure of the ribosome-component of nRNAg. The cryo-EM reveals that predominant particles in nRNAg are 80S ribosomes, resembling the pre-translocation state where tRNA’s are in the hybrid A/P and P/E site. We also describe a new kind of principal motion of the ribosome, which we call the rocking motion.

In situ single particle classification reveals distinct 60S maturation intermediates in cells.

Previously, we showed that high-resolution template matching can localize ribosomes in two-dimensional electron cryo-microscopy (cryo-EM) images of untilted Mycoplasma pneumoniae cells with high precision (Lucas et al., 2021). Here, we show that comparing the signal-to-noise ratio (SNR) observed with 2DTM using different templates relative to the same cellular target can correct for local variation in noise and differentiate related complexes in focused ion beam (FIB)-milled cell sections. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the Saccharomyces cerevisiae cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. 2DTM can distinguish related molecular populations without the need to generate 3D reconstructions from the data to be classified, permitting classification even when only a few target particles exist in a cell.

N6-methyladenosine (m6A) in 18S rRNA promotes fatty acid metabolism and oncogenic transformation.

Aberrant RNA modifications lead to dysregulated gene expression and cancer progression. Ribosomal RNA (rRNA) accounts for more than 80% of a cell's total RNA, but the functions and molecular mechanisms underlying rRNA modifications in cancers are poorly understood. Here, we show that the 18S rRNA N6-methyladenosine (m6A) methyltransferase complex METTL5-TRMT112 is upregulated in various cancer types and correlated with poor prognosis. In addition, we demonstrate the critical functions of METTL5 in promoting hepatocellular carcinoma (HCC) tumorigenesis in vitro and in mouse models. Mechanistically, depletion of METTL5-mediated 18S rRNA m6A modification results in impaired 80S ribosome assembly and decreased translation of mRNAs involved in fatty acid metabolism. We further reveal that ACSL4 mediates the function of METTL5 on fatty acid metabolism and HCC progression, and targeting ACSL4 and METTL5 synergistically inhibits HCC tumorigenesis in vivo. Our study uncovers mechanistic insights underlying mRNA translation control and HCC tumorigenesis through lipid metabolism remodeling and provides a molecular basis for the development of therapeutic strategies for HCC treatment.

The Quality Control of Midecamycin and the Predictive Demarcation between Its Impurities and Components

Midecamycin is a 16-membered macrolide antibiotic. It can inhibit the synthesis of bacterial proteins by blocking up the activity of peptidyl transferase in the 50S ribosome. We used high-resolution mass spectrometry to analyze midecamycin, and quantitatively analyzed of each component of midecamycin produced by 18 different companies. The developed methods were validated by assessing linearity, limit of quantitation (LOQ), accuracy, precision, and robustness. Good separations were achieved for all components. Ten components of midecamycin were identified, and the contents of these components were determined in midecamycin produced by different companies. The demarcation between impurities and components of midecamycin was not clear. A ligand-docking model was used for predicting the impurities and components of midecamycin. Components and impurities were docked with the target. The results reported in this article may be important for quality control and the predictive demarcation between impurities and components of midecamycin.

In vitro and invivo detection of microbial gene expression in bioactivated scaffolds seeded with cyanobacteria.

Dermal replacement materials bioactivated with cyanobacteria have shown promising potential for wound regeneration. To date, extraction of cyanobacteria RNA from seeded scaffolds has not been described. The aim of this study was to develop a method to isolate total RNA from bioactivated scaffolds and to propose a new approach in determining living bacteria based on real-time PCR. Transgenic Synechococcus sp. PCC 7002 (tSyn7002) were seeded in liquid cultures or scaffolds for dermal regeneration invitro and invivo for 7 days. RNA was extracted with a 260/280 ratio of ≥2. The small subunit of the 30S ribosome in prokaryotes (16S) and RNAse P protein (rnpA) were validated as reference transcripts for PCR analysis. Gene expression patterns differed invitro and invivo. Expression of 16S was significantly upregulated in scaffolds invitro, as compared to liquid cultures, whilst rnpA expression was comparable. In vivo, both 16S and rnpA showed reduced expression compared to invitro (16S: invivo Ct value 13.21 ± 0.32, invitro 12.44 ± 0.42; rnpA invivo Ct value 19.87 ± 0.41, invitro 17.75 ± 1.41). Overall, the results demonstrate rnpA and 16S expression after 7 days of implantation invitro and invivo, proving the presence of living bacteria embedded in scaffolds using qPCR.

Design, synthesis, biological evaluation and molecular docking studies of novel pleuromutilin derivatives containing nitrogen heterocycle and alkylamine groups

A series of pleuromutilin derivatives containing alkylamine and nitrogen heterocycle groups were designed and synthesised under mild conditions. The in vitro antibacterial activity of these semisynthetic derivatives against four strains of Staphylococcus aureus (MRSA ATCC 43300, S.aureus ATCC 29213, S.aureus AD3, and S.aureus 144) were evaluated by the broth dilution method. Compound 13 was found to have excellent antibacterial activity against MRSA (MIC = 0.0625 μg/mL). Furthermore, compound 13 was further studied by the time-killing kinetics and the post-antibiotic effect approach. In the mouse thigh infection model, compound 13 exhibited superior antibacterial efficacy than that of tiamulin. Meanwhile, compound 13 showed a lower inhibitory effect than that of tiamulin on RAW264.7 and 16HBE cells at the concentration of 10 μg/mL. Molecular docking study revealed that compound 13 can effectively bind to the active site of the 50S ribosome (the binding free energy = −9.66 kcal/mol).

Selective footprinting of 40S and 80S ribosome subpopulations (Sel-TCP-seq) to study translation and its control.

Multiple aspects of mRNA translation are subject to regulation. Here we present a ribosome footprinting protocol to determine the location and composition of 40S and 80S ribosome complexes on endogenous mRNAs transcriptome-wide in vivo in yeast and mammalian cells. We present an extension of the translation complex profiling (TCP-seq) protocol, originally developed in yeast, by including an immunoprecipitation step to assay the location of both 40S and 80S ribosome complexes containing proteins of interest. This yields information on where along mRNAs the ribosome-bound protein of interest joins the ribosome to act, and where it leaves again, thereby monitoring the sequential steps of translation and the roles of various translation factors therein. Rapid fixation of live cells ensures the integrity of all translation complexes bound to mRNA at native positions. Two procedures are described, differing mainly in the fixation conditions and the library preparation. Depending on the research question, either procedure offers advantages. Execution of a Sel-TCP-seq experiment takes 5-10 working days, and initial data analysis can be completed within 2 days.

Modulating co-translational protein folding by rational design and ribosome engineering

Co-translational folding is a fundamental process for the efficient biosynthesis of nascent polypeptides that emerge through the ribosome exit tunnel. To understand how this process is modulated by the shape and surface of the narrow tunnel, we have rationally engineered three exit tunnel protein loops (uL22, uL23 and uL24) of the 70S ribosome by CRISPR/Cas9 gene editing, and studied the co-translational folding of an immunoglobulin-like filamin domain (FLN5). Our thermodynamics measurements employing 19F/15N/methyl-TROSY NMR spectroscopy together with cryo-EM and molecular dynamics simulations reveal how the variations in the lengths of the loops present across species exert their distinct effects on the free energy of FLN5 folding. A concerted interplay of the uL23 and uL24 loops is sufficient to alter co-translational folding energetics, which we highlight by the opposite folding outcomes resulting from their extensions. These subtle modulations occur through a combination of the steric effects relating to the shape of the tunnel, the dynamic interactions between the ribosome surface and the unfolded nascent chain, and its altered exit pathway within the vestibule. These results illustrate the role of the exit tunnel structure in co-translational folding, and provide principles for how to remodel it to elicit a desired folding outcome.