Studying the Structural Organization of Polyribosomes with Alexander S. Spirin.

"Would it be possible to analyze molecular mechanisms and structural organisation of polyribosome assemblies using cryo electron tomography?" - we asked through a longstanding collaboration between my research group and that of Alexander S. Spirin. Indeed, it was: we found that double-row polyribosomes can have both circular and linear arrangements of their mRNA [Afonina, Z. A., et al. (2013) Biochemistry (Moscow)], we figured out how eukaryotic ribosomes assemble on an mRNA to form supramolecular left-handed helices [Myasnikov, A. G., et al. (2014) Nat. Commun.], that the circularization of polyribosomes is poly-A and cap-independent [Afonina, Z. A., et al. (2014) Nucleic Acids Res.], and that intermediary polyribosomes with open structures exist after a transition from a juvenile phase to strongly translating polysomes of medium size [Afonina, Z. A., et al. (2015) Nucleic Acids Res.] until they form densely packed helical structures with reduced activity. Our joint fruitful exchanges, hence, led to major advances in the field, which are reviewed here from a personal and historical perspective in memory of Alexander S. Spirin.

The oncomicropeptide APPLE promotes hematopoietic malignancy by enhancing translation initiation.

Initiation is the rate-limiting step in translation, and its dysregulation is vital for carcinogenesis, including hematopoietic malignancy. Thus, discovery of novel translation initiation regulators may provide promising therapeutic targets. Here, combining Ribo-seq, mass spectrometry, and RNA-seq datasets, we discovered an oncomicropeptide, APPLE (a peptide located in ER), encoded by a non-coding RNA transcript in acute myeloid leukemia (AML). APPLE is overexpressed in various subtypes of AML and confers a poor prognosis. The micropeptide is enriched in ribosomes and regulates the initiation step to enhance translation and to maintain high rates of oncoprotein synthesis. Mechanically, APPLE promotes PABPC1-eIF4G interaction and facilitates mRNA circularization and eIF4F initiation complex assembly to support a specific pro-cancer translation program. Targeting APPLE exhibited broad anti-cancer effects in vitro and in vivo. This study not only reports a previously unknown function of micropeptides but also provides new opportunities for targeting the translation machinery in cancer cells.

Cotranslational prolyl hydroxylation is essential for flavivirus biogenesis.

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.

Stochastic microswimming model of ribosome motion on the polysome.

In this work we assume that the ribosome propels itself during the translocation step of the translation process of protein synthesis by running a cycle of stochastically generated conformational changes involving its two subunits. This cycle includes only two experimentally found ribosome shape changes. The main result is an analytic expression for ribosome's average swimming speed on a polysome, where the ribosome is in the presence of other ribosomes. Relevant geometric parameters of ribosome deformations are calculated first by solving a deterministic problem where the ribosome runs a cycle of prescribed conformational changes. The method of reflections and pairwise additivity are used to obtain the stresses and forces needed to apply the multiparticle reciprocal theorem. Ribosome's average velocity when it runs the corresponding stochastic cycle of deformations is calculated assuming independence among the conformational cycles of different ribosomes on the polysome. The results obtained show that swimming in tandem on the polysome allows the ribosome to reach any typical subcellular speed with deformations whose amplitude is of a smaller size than when it swims alone in the fluid. Also, the flow organized by its swimming stroke becomes more determinant for its motion than random diffusion, compared to the solitary ribosome.

Polysomes Bypass a 50-Nucleotide Coding Gap Less Efficiently Than Monosomes Due to Attenuation of a 5' mRNA Stem-Loop and Enhanced Drop-off.

Efficient translational bypassing of a 50-nt non-coding gap in a phage T4 topoisomerase subunit gene (gp60) requires several recoding signals. Here we investigate the function of the mRNA stem-loop 5' of the take-off codon, as well as the importance of ribosome loading density on the mRNA for efficient bypassing. We show that polysomes are less efficient at mediating bypassing than monosomes, both in vitro and in vivo, due to their preventing formation of a stem-loop 5' of the take-off codon and allowing greater peptidyl-tRNA drop off. A ribosome profiling analysis of phage T4-infected Escherichia coli yielded protected mRNA fragments within the normal size range derived from ribosomes stalled at the take-off codon. However, ribosomes at this position also yielded some 53-nucleotide fragments, 16 longer. These were due to protection of the nucleotides that form the 5' stem-loop. NMR shows that the 5' stem-loop is highly dynamic. The importance of different nucleotides in the 5' stem-loop is revealed by mutagenesis studies. These data highlight the significance of the 5' stem-loop for the 50-nt bypassing and further enhance appreciation of relevance of the extent of ribosome loading for recoding.

Multiple links between 5-methylcytosine content of mRNA and translation.

BACKGROUND: 5-Methylcytosine (m5C) is a prevalent base modification in tRNA and rRNA but it also occurs more broadly in the transcriptome, including in mRNA, where it serves incompletely understood molecular functions. In pursuit of potential links of m5C with mRNA translation, we performed polysome profiling of human HeLa cell lysates and subjected RNA from resultant fractions to efficient bisulfite conversion followed by RNA sequencing (bsRNA-seq). Bioinformatic filters for rigorous site calling were devised to reduce technical noise. RESULTS: We obtained ~ 1000 candidate m5C sites in the wider transcriptome, most of which were found in mRNA. Multiple novel sites were validated by amplicon-specific bsRNA-seq in independent samples of either human HeLa, LNCaP and PrEC cells. Furthermore, RNAi-mediated depletion of either the NSUN2 or TRDMT1 m5C:RNA methyltransferases showed a clear dependence on NSUN2 for the majority of tested sites in both mRNAs and noncoding RNAs. Candidate m5C sites in mRNAs are enriched in 5'UTRs and near start codons and are embedded in a local context reminiscent of the NSUN2-dependent m5C sites found in the variable loop of tRNA. Analysing mRNA sites across the polysome profile revealed that modification levels, at bulk and for many individual sites, were inversely correlated with ribosome association. CONCLUSIONS: Our findings emphasise the major role of NSUN2 in placing the m5C mark transcriptome-wide. We further present evidence that substantiates a functional interdependence of cytosine methylation level with mRNA translation. Additionally, we identify several compelling candidate sites for future mechanistic analysis.

Polysome Profiling and Metabolic Labeling Methods to Measure Translation in Trypanosoma brucei.

The amount of a protein that is made in a cell is determined not only by the corresponding mRNA level but also by the efficiency with which the mRNA is translated. Very powerful transcriptome-wide methods are available to analyze both the density of ribosomes on each mRNA and the rate at which polypeptides are elongated. However, for many research questions, simpler, less expensive methods are more suitable. Here we describe two methods to assess the general translation status of cells: polysome profiling by sucrose density gradient centrifugation and metabolic labeling using radioactive amino acids. Both methods can also be used to examine translation of individual mRNAs.

Tetrapeptide 60-63 of human ribosomal protein uS3 is crucial for translation initiation.

Conserved ribosomal protein uS3 contains a decapeptide fragment in positions 55-64 (human numbering), which has a very specific ability to cross-link to various RNA derivatives bearing aldehyde groups, likely provided by K62. It has been shown that during translation in the cell-free protein-synthesizing system, uS3 becomes accessible for such cross-linking only after eIF3j leaves the mRNA binding channel of the 40S ribosomal subunit. We studied the functional role of K62 and its nearest neighbors in the ribosomal assembly and translation with the use of HEK293T-derived cell cultures capable of producing FLAG-tagged uS3 (uS3FLAG) or its mutant form with amino acid residues at positions 60-63 replaced with alanines. Analysis of polysome profiles from the respective cells and cytosol lysates showed that the mutation significantly affected the uS3 ability to participate in the assembly of 40S subunits, but it was not essential for their maturation and did not prevent the binding of mRNAs to 40S subunits during translation initiation. The most striking effect of the replacement of amino acid residues in the above uS3 positions was that it almost completely deprived the 40S subunits of their ability to form 80S ribosomes, suggesting that the 48S pre-initiation complexes assembled on these subunits were defective in the binding of 60S subunits. Thus, our results revealed the previously unknown crucial role of the uS3 tetrapeptide 60GEKG63 in translation initiation related to maintaining the proper structure of the 48S complex, most likely via the prevention of premature mRNA loading into the ribosomal channel.

mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis.

N6-methyladenosine (m6A) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered m6A homeostasis is linked to cancer1-5. m6A modification is catalysed by METTL3 and enriched in the 3' untranslated region of a large subset of mRNAs at sites close to the stop codon5. METTL3 can promote translation but the mechanism and relevance of this process remain unknown1. 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 m6A-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.

Functional mapping of the E. coli translational machinery using single-molecule tracking.

The organization of the chromosomal DNA and ribosomes in living Escherichia coli is compared under two growth conditions: 'fast' (50 min doubling time) and 'slow' (147 min doubling time). Superresolution fluorescence microscopy reveals strong DNA-ribosome segregation in both cases. In both fast and slow growth, free ribosomal subunits evidently must circulate between the nucleoid (where they initiate co-transcriptional translation) and ribosome-rich regions (where most translation occurs). Single-molecule diffusive behavior dissects the ribosome copies into translating 70S polysomes and free 30S subunits, providing separate spatial distributions for each. In slow growth, ~21,000 total 30S copies/cell comprise ~65% translating 70S ribosomes and ~35% free 30S subunits. The ratio of 70S ribosomes to free 30S subunits is ~2.5 outside the nucleoid and ~0.50 inside the nucleoid. This new level of quantitative detail may motivate development of comprehensive, three-dimensional reaction-diffusion models of ribosome, DNA, mRNA and RNAP spatial distributions and dynamics within the E. coli cytoplasm.

Efficient analysis of mammalian polysomes in cells and tissues using Ribo Mega-SEC.

We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.

The Institute of Protein Research of the Russian Academy of Sciences Is 50 Years Old.

Here I introduce collection of review articles written by members of the Institute of Protein Research of the Russian Academy of Sciences. This collection commemorates the 50th anniversary of the Institute. The review articles cover a broad range of problems concerning the spatial structure of protein molecules, including the state of the molten globule, protein-RNA interactions, polysome and ribosome structure, the molecular colony method, and the original methods for studying the structure of proteins. Several of the reviews consider the practical use of knowledge about the structure of proteins and protein polymers. They reflect both the long experience of the authors and contemporary scientific data.

Three-Dimensional Organization of Polyribosomes - A Modern Approach.

Polyribosomes in cells usually have a certain structural organization whose significance has not yet been elucidated. The development of cryo electron tomography has provided a new approach to study polyribosome structure. New data confirm or correct observations made earlier by classical techniques of electron microscopy. The existence of circular and linear (zigzag) topology of polyribosomes was confirmed, and their relationship with the frequently observed two-row forms was clarified. Contacts between ribosomes have been identified in densely packed three-dimensional helical polyribosomes. At the same time, modern cell-free translation systems have opened the possibility of investigating polyribosomes on mRNA of a given structure to elucidate the mechanism of polyribosome structure formation, especially of circular polyribosomes. There is an increasing amount of data supporting the idea of interdependence between polyribosome structure and their translational activity. Moreover, participation of polyribosomes in mRNA transport and localization of protein synthesis in the cell has been shown. Improvement of the resolution and the development of the cryo electron tomography technique for the analysis of polyribosomes in situ will enable further progress in understanding the process of protein synthesis in cells.

Preparation of polysomal fractions from mouse brain synaptoneurosomes and analysis of polysomal-bound mRNAs.

BACKGROUND: Here we describe a detailed, reliable protocol for isolation of polysomal fractions from mouse brain synaptoneurosomes. This method is an important tool to study local protein synthesis in neurons. NEW METHOD: We combined rapid preparation of synaptoneurosomes by filtration with polysome profiling. We provide a detailed protocol highlighting difficulties and critical steps of: i) preparation of synaptoneurosomes; ii) polyribosome fractionation from synaptoneurosomes; iii) extraction of proteins and RNA from sucrose gradient fractions. RESULTS: and Comparison with Existing Methods We fractionated polyribosomes from synaptoneurosomes and detected the association of Mmp9, Camk2a and Stx1B mRNA with polysomes in the unstimulated conditions. Synaptic stimulation led to increased levels of Mmp9 and Camk2a mRNA in the heavy polysomal fractions. We compared our protocol with existing methods CONCLUSIONS: We have developed a reliable, effective method to prepare polyribosomal fractions from synaptoneurosomes to study polyribosomal binding of mRNAs as an aspect of synaptic translation in vitro.

Isolation of Polysomal RNA for Analyzing Stress-Responsive Genes Regulated at the Translational Level in Plants.

Alteration of gene expression is an essential mechanism, which allows plants to respond and adapt to adverse environmental conditions. Transcriptome and proteome analyses in plants exposed to abiotic stresses revealed that protein levels are not correlated with the changes in corresponding mRNAs, indicating regulation at translational level is another major regulator for gene expression. Analysis of translatome, which refers to all mRNAs associated with ribosomes, thus has the potential to bridge the gap between transcriptome and proteome. Polysomal RNA profiling and recently developed ribosome profiling (Ribo-seq) are two main methods for translatome analysis at global level. Here, we describe the classical procedure for polysomal RNA isolation by sucrose gradient ultracentrifugation followed by highthroughput RNA-seq to identify genes regulated at translational level. Polysomal RNA can be further used for a variety of downstream applications including Northern blot analysis, qRT-PCR, RNase protection assay, and microarray-based gene expression profiling.

Detection of Argonaute 1 Association with Polysomes in Arabidopsis thaliana.

Argonaute (AGO) proteins play a key role in RNA silencing mechanisms. RNA silencing affects both RNA degradation and translation. The characterization of translation-associated RNA silencing mechanisms and components often requires polysome isolation and analysis. In this chapter, we describe the identification of AGO1 association with polysomes through polysome fractionation on sucrose gradient, preparation of proteins by filtration and concentration, and immunoblotting.

Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis.

mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilise available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localised to the translationally active heavy polysomes. The highest titre cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5' untranslated regions that might be utilised to enhance recombinant transcript translation.

Purification, identification, and functional analysis of polysomes from the human pathogen Staphylococcus aureus.

Polysomes are macromolecular complexes made up of multiple ribosomes simultaneously translating a single mRNA into polypeptide chains. Together, the cellular mRNAs translated in this way are referred to 'translatome.' Translation determines a cell's overall gene expression profile. Studying translatome leads to a better understanding of the translational machinery and of its complex regulatory pathways. Given its fundamental role in cell homeostasis and division, bacterial translation is an important target for antibiotics. However, there are no detailed protocols for polysome purification from Staphylococcus aureus, the human pathogen responsible for the majority of multi-drug resistance issues. We therefore developed methods for the isolation of active polysomes, ribosomes, and ribosomal subunits, examining the purity and quality of each fraction and monitoring polysomal activity during protein synthesis. These steps are mandatory for the use of purified S. aureus polysomes and ribosomes for structural studies or for genome-scale analysis of most translated mRNAs.

The Histone Deacetylase Gene Rpd3 Is Required for Starvation Stress Resistance.

Epigenetic regulation in starvation is important but not fully understood yet. Here we identified the Rpd3 gene, a Drosophila homolog of histone deacetylase 1, as a critical epigenetic regulator for acquiring starvation stress resistance. Immunostaining analyses of Drosophila fat body revealed that the subcellular localization and levels of Rpd3 dynamically changed responding to starvation stress. In response to starvation stress, the level of Rpd3 rapidly increased, and it accumulated in the nucleolus in what appeared to be foci. These observations suggest that Rpd3 plays a role in regulation of rRNA synthesis in the nucleolus. The RT-qPCR and ChIP-qPCR analyses clarified that Rpd3 binds to the genomic region containing the rRNA promoters and activates rRNA synthesis in response to starvation stress. Polysome analyses revealed that the amount of polysomes was decreased in Rpd3 knockdown flies under starvation stress compared with the control flies. Since the autophagy-related proteins are known to be starvation stress tolerance proteins, we examined autophagy activity, and it was reduced in Rpd3 knockdown flies. Taken together, we conclude that Rpd3 accumulates in the nucleolus in the early stage of starvation, upregulates rRNA synthesis, maintains the polysome amount for translation, and finally increases stress tolerance proteins, such as autophagy-related proteins, to acquire starvation stress resistance.

StructMap: Elastic Distance Analysis of Electron Microscopy Maps for Studying Conformational Changes.

Single-particle electron microscopy (EM) has been shown to be very powerful for studying structures and associated conformational changes of macromolecular complexes. In the context of analyzing conformational changes of complexes, distinct EM density maps obtained by image analysis and three-dimensional (3D) reconstruction are usually analyzed in 3D for interpretation of structural differences. However, graphic visualization of these differences based on a quantitative analysis of elastic transformations (deformations) among density maps has not been done yet due to a lack of appropriate methods. Here, we present an approach that allows such visualization. This approach is based on statistical analysis of distances among elastically aligned pairs of EM maps (one map is deformed to fit the other map), and results in visualizing EM maps as points in a lower-dimensional distance space. The distances among points in the new space can be analyzed in terms of clusters or trajectories of points related to potential conformational changes. The results of the method are shown with synthetic and experimental EM maps at different resolutions.