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Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells 1 . Here we use advances in cryo-electron tomography and sub-tomogram analysis 2 , 3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae . To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M.   pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes 4 . By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells. Cryo-electron tomography is used to reveal the structural dynamics and functional diversity of translating ribosomes in Mycoplasma pneumoniae , providing insight into the translation elongation cycle inside cells and how it is reshaped by antibiotics.

N 6 -methyladenosine (m 6 A) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered m 6 A homeostasis is linked to cancer 1 – 5 . m 6 A modification is catalysed by METTL3 and enriched in the 3′ untranslated region of a large subset of mRNAs at sites close to the stop codon 5 . METTL3 can promote translation but the mechanism and relevance of this process remain unknown 1 . Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5′ cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs—including bromodomain-containing protein 4—that is also m 6 A-modified in human primary lung tumours. The METTL3–eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3–eIF3h as a potential therapeutic target for patients with cancer. METTL3, the enzyme responsible for m 6 A modification, influences translation by interacting with eIF3h to mediate looping between the regions near the stop codon and 5′ cap of mRNA.

Background Polysome profiling is a widespread technique to study mRNA translation. After separation of cellular particles by ultracentrifugation on a sucrose-density gradient, a UV absorbance profile is recorded during elution, which mostly reflects RNA content and shows distinct peaks for ribosomal subunits, monosomes and polysomes with increasing number of ribosomes. This profile can be used to assess global translational activity, or to reveal changes in ribosome biogenesis and translation elongation. In addition, it is also possible to measure the association of fluorescently tagged proteins with ribosomal subunits or polysomes. Alignment and quantification of polysome profiles usually relies on spreadsheet programs, custom R/Python scripts or commercial software. Results With QuAPPro, we present the first interactive web app that allows quantification and alignment of polysome profiles, independently of the device or software that was used to generate the profiles. QuAPPro was written in R, with a graphical user interface implemented in R shiny. It supports interactive visualization and analysis of polysome profiles, including profile smoothing, baseline selection, alignment along a defined point on the x-axis, quantification of profile subsections and deconvolution for resolving individual peaks. Fluorescence profiles can be aligned and quantified in parallel. Finally, quantification results can be summarized and visualized as bar plots. Every interactive plot can be exported directly in a publication-ready format. Conclusions This user-friendly tool does not only speed up the analysis of polysome profiles but also facilitates reproducibility and documentation of the process, without the need for programming abilities or commercial software.

Single-stranded circular RNAs (circRNAs), generated through ‘backsplicing’, occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. Virus-derived circRNAs have recently been described in gamma-herpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate circRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N 6 -methyladenosine (m 6 A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV. The authors identify circular RNAs (circRNA) from human papillomavirus and show that circRNA-encoded E7 contributes to cancer cell growth in vitro and in tumor xenografts. Furthermore, circE7 is present in TCGA RNA-Seq data from HPV-positive cancers.

Local translation allows for a spatial control of gene expression. Here, we use high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging reveals active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysome transport mediated by nascent proteins. Centrosomes function as microtubule organizing centers where several mRNAs accumulate. By employing high-throughput single molecule FISH screening, the authors discover that 8 human mRNAs localize to centrosomes with unique cell cycle dependent patterns using an active polysome targeting mechanism.

In the absence of extensive transcription control mechanisms the pathogenic parasite Trypanosoma brucei crucially depends on translation regulation to orchestrate gene expression. However, molecular insight into regulating protein biosynthesis is sparse. Here we analyze the small non-coding RNA (ncRNA) interactome of ribosomes in T. brucei during different growth conditions and life stages. Ribosome-associated ncRNAs have recently been recognized as unprecedented regulators of ribosome functions. Our data show that the tRNA Thr 3´half is produced during nutrient deprivation and becomes one of the most abundant tRNA-derived RNA fragments (tdRs). tRNA Thr halves associate with ribosomes and polysomes and stimulate translation by facilitating mRNA loading during stress recovery once starvation conditions ceased. Blocking or depleting the endogenous tRNA Thr halves mitigates this stimulatory effect both in vivo and in vitro . T. brucei and its close relatives lack the well-described mammalian enzymes for tRNA half processing, thus hinting at a unique tdR biogenesis in these parasites. Trypanosoma brucei mainly relies on translational regulation to adjust gene expression, but details are unclear. Here the authors show that, under stress conditions, tRNA Thr half level increases, associates with ribosomes and polysomes, and stimulates protein synthesis by facilitating mRNA loading.

Protein synthesis is crucial for the maintenance of long-term memory-related synaptic plasticity. The cytoplasmic polyadenylation element-binding protein 3 (CPEB3) regulates the translation of several mRNAs important for long-term synaptic plasticity in the hippocampus. In previous studies, we found that the oligomerization and activity of CPEB3 are controlled by small ubiquitin-like modifier (SUMO)ylation. In the basal state, CPEB3 is SUMOylated; it is soluble and acts as a repressor of translation. Following neuronal stimulation, CPEB3 is de-SUMOylated; it now forms oligomers that are converted into an active form that promotes the translation of target mRNAs. To better understand how CPEB3 regulates the translation of its mRNA targets, we have examined CPEB3 subcellular localization. We found that basal, repressive CPEB3 is localized to membraneless cytoplasmic processing bodies (P bodies), subcellular compartments that are enriched in translationally repressed mRNA. This basal state is affected by the SUMOylation state of CPEB3. After stimulation, CPEB3 is recruited into polysomes, thus promoting the translation of its target mRNAs. Interestingly, when we examined CPEB3 recombinant protein in vitro, we found that CPEB3 phase separates when SUMOylated and binds to a specific mRNA target. These findings suggest a model whereby SUMO regulates the distribution, oligomerization, and activity of oligomeric CPEB3, a critical player in the persistence of memory.

Comprehensive in situ structures of macromolecules can transform our understanding of biology and advance human health. Here, we map protein synthesis inside human cells in detail by combining automated cryo-focused ion beam (FIB) milling and in situ single-particle cryo electron microscopy (cryo-EM). With this in situ cryo-EM approach, we resolved a 2.2 Å consensus structure of the human 80S ribosome and unveiled 23 functional states, nearly all better than 3 Å resolution. Compared to in vitro studies, we observed variations in ribosome structures, distinct environments of ion and polyamine binding, and associated proteins such as EDF1 and NAC β that are typically not enriched with purified ribosomes. We also detected additional peptide-related density features on the ribosome and visualized ribosome–ribosome interactions in helical polysomes. Finally, high-resolution structures from cells treated with homoharringtonine and cycloheximide revealed a distinct translational landscape and a spermidine that interacts with cycloheximide at the E site, one of the numerous polyamines that also bind native ribosomes. These results underscore the value of high-resolution in situ studies in the native environment. Understanding protein synthesis in its cellular context is essential. Here, authors apply in situ cryo-EM to reveal the human ribosome at 2.2 Å resolution, capture 23 states, and uncover drug-specific translation features in native cells.

Like all other cells, neurons use different proteins to process information and respond to stimuli. To meet the huge demands for new proteins in their large and complex cell volume, neurons have moved the protein templates—messenger RNAs (mRNAs)—and the protein synthesis machines—ribosomes—out to synapses to make proteins locally. During protein synthesis, multiple ribosomes can form a structure known as a polysome, which produces multiple protein copies from a single mRNA. Working in rodent preparations, Biever et al. found that solitary, mRNA-associated ribosomes, or monosomes, are a substantial source of proteins in neuronal processes. Many synaptic proteins are made on single ribosomes, which may solve the problem of limited space in tiny synaptic compartments. Science , this issue p. eaay4991 Protein translation on monosomes rather than polysomes contributes to the local neuronal proteome in rodents. To accommodate their complex morphology, neurons localize messenger RNAs (mRNAs) and ribosomes near synapses to produce proteins locally. However, a relative paucity of polysomes (considered the active sites of translation) detected in electron micrographs of neuronal processes has suggested a limited capacity for local protein synthesis. In this study, we used polysome profiling together with ribosome footprinting of microdissected rodent synaptic regions to reveal a surprisingly high number of dendritic and/or axonal transcripts preferentially associated with monosomes (single ribosomes). Furthermore, the neuronal monosomes were in the process of active protein synthesis. Most mRNAs showed a similar translational status in the cell bodies and neurites, but some transcripts exhibited differential ribosome occupancy in the compartments. Monosome-preferring transcripts often encoded high-abundance synaptic proteins. Thus, monosome translation contributes to the local neuronal proteome.

One of the morphological hallmarks of terminally differentiated secretory cells is highly proliferated membrane of the rough endoplasmic reticulum (ER), but the molecular basis for the high rate of protein biosynthesis in these cells remains poorly documented. An important aspect of ER translational control is the molecular mechanism that supports efficient use of targeted mRNAs in polyribosomes. Here, we identify an enhancement system for ER translation promoted by p180, an integral ER membrane protein we previously reported as an essential factor for the assembly of ER polyribosomes. We provide evidence that association of target mRNAs with p180 is critical for efficient translation, and that SF3b4, an RNA-binding protein in the splicing factor SF3b, functions as a cofactor for p180 at the ER and plays a key role in enhanced translation of secretory proteins. A cis-element in the 5′ untranslated region of collagen and fibronectin genes is important to increase translational efficiency in the presence of p180 and SF3b4. These data demonstrate that a unique system comprising a p180–SF3b4–mRNA complex facilitates the selective assembly of polyribosomes on the ER.