Ribosome Profiling and RNA Sequencing Reveal Genome-Wide Cellular Translation and Transcription Regulation Under Osmotic Stress in Lactobacillus rhamnosus ATCC 53103

To determine whether osmotic pressure affects the translation efficiency of Lactobacillus rhamnosus, the ribosome profiling assay was performed to analyze the changes in translation efficiency in L. rhamnosus ATCC 53103. Under osmotic stress, differentially expressed genes (DEGs) involved in fatty acid biosynthesis and metabolism, ribosome, and purine metabolism pathways were co-regulated with consistent expression direction at translation and transcription levels. DEGs involved in the biosynthesis of phenylalanine, tyrosine, and tryptophan, and the phosphotransferase system pathways also were co-regulated at translation and transcription levels, while they showed opposite expression direction at two levels. Moreover, DEGs involved in the two-component system, amino acid metabolism, and pyruvate metabolism pathways were only regulated at the transcription level. And DEGs involved in fructose and mannose metabolism were only regulated at the translation level. The translation efficiency of DEGs involved in the biosynthesis of amino acids was downregulated while in quorum sensing and PTS pathways was upregulated. In addition, the ribosome footprints accumulated in open reading frame regions resulted in impaired translation initiation and elongation under osmotic stress. In summary, L. rhamnosus ATCC 53103 could respond to osmotic stress by translation regulation and control the balance between survival and growth of cells by transcription and translation.

Polyamines mediate enterovirus attachment directly and indirectly through cellular heparan sulfate synthesis.

Productive viral infection begins with attachment to a susceptible cell, and viruses have evolved complex mechanisms to attach to and subsequently enter cells. Prior to engagement with a cellular receptor, viruses frequently interact with nonspecific attachment factors that can facilitate virus-receptor interactions and viral entry. Polyamines, small positively-charged molecules abundant in mammalian cells, mediate viral attachment, though the mechanism was not fully understood. Using the Coxsackievirus B3 (CVB3) enterovirus model system, we show that polyamines mediate viral attachment both directly and indirectly. The polyamine putrescine specifically enhances viral attachment to cells depleted of polyamines. Putrescine's positive charge mediates its ability to enhance viral attachment, and polyamine analogs are less efficient at mediating viral attachment. In addition to this direct role of polyamines in attachment, polyamines facilitate the cellular expression of heparan sulfates, negatively-charged molecules found on the cell surface. In polyamine-depleted cells, heparan sulfates are depleted from the surface of cells, resulting in reduced viral attachment. We find that this is due to polyamines' role in the process of hypusination of eukaryotic initiation factor 5A, which facilitates cellular translation. These data highlight the important role of polyamines in mediating cellular attachment, as well as their function in facilitating cellular heparan sulfate synthesis.

Ribosomal RNA 2'-O-methylations regulate translation by impacting ribosome dynamics

Protein synthesis by ribosomes is critically important for gene expression in all cells. The ribosomal RNAs (rRNAs) are marked by numerous chemical modifications. An abundant group of rRNA modifications, present in all domains of life, is 2'-O-methylation guided by box C/D small nucleolar RNAs (snoRNAs) which are part of small ribonucleoprotein complexes (snoRNPs). Although 2'-O-methylations are required for proper production of ribosomes, the mechanisms by which these modifications contribute to translation have remained elusive. Here, we show that a change in box C/D snoRNP biogenesis in actively growing yeast cells results in the production of hypo 2'-O-methylated ribosomes with distinct translational properties. Using RiboMeth-Seq for the quantitative analysis of 2'-O methylations, we identify site-specific perturbations of the rRNA 2'-O-methylation pattern and uncover sites that are not required for ribosome production under normal conditions. Characterization of the hypo 2'-O-methylated ribosomes reveals significant translational fidelity defects including frameshifting and near-cognate start codon selection. Using rRNA structural probing, we show that hypo 2'-O-methylation affects the inherent dynamics of the ribosomal subunits and impacts the binding of translation factor eIF1 thereby causing translational defects. Our data reveal an unforeseen spectrum of 2'-O-methylation heterogeneity in yeast rRNA and suggest a significant role for rRNA 2'-O-methylation in regulating cellular translation by controlling ribosome dynamics and ligand binding.

Mechanisms of translation inhibition and suppression of Stress Granule formation by cisplatin

Platinum-based antineoplastic drugs, such as cisplatin, are commonly used to induce tumor cell death. Cisplatin is believed to induce apoptosis as a result of cisplatin-DNA adducts that inhibit DNA and RNA synthesis. Although idea that DNA damage underlines anti-proliferative effects of cisplatin is dominant in cancer research, there is a poor correlation between the degree of the cell sensitivity to cisplatin and the extent of DNA platination. Here, we propose a novel mechanism of cisplatin-mediated cytotoxicity. We show that cisplatin suppresses formation of Stress Granules (SGs), pro-survival RNA granules with multiple roles in cellular metabolism. Mechanistically, cisplatin inhibits cellular translation to promote disassembly of polysomes and aggregation of ribosomal subunits. As SGs are in equilibrium with polysomes, cisplatin-induced shift towards ribosomal aggregation suppresses SG formation and promotes cellular death. Our data also explain nephrotoxic, neurotoxic and ototoxic effects of cisplatin treatment.

Context-specific action of macrolide antibiotics on the eukaryotic ribosome

Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

Phylogeny Reveals Novel HipA-Homologous Kinase Families and Toxin – Antitoxin Gene Organizations

Toxin – Antitoxin modules function in the genetic stability of mobile genetic elements, bacteriophage defense, and antibiotic tolerance. A gain-of-function mutation of the Escherichia coli K-12 hipBA module can induce antibiotic tolerance in a subpopulation of bacterial cells, a phenomenon known as persistence. HipA is a Ser/Thr kinase that phosphorylates and inactivates glutamyl tRNA synthetase, inhibiting cellular translation and inducing the stringent response. Additional characterized HipA homologues include HipT from pathogenic E. coli O127 and YjjJ of E. coli K-12, which are encoded by tri-cistronic hipBST and monocistronic operons, respectively. The apparent diversity of HipA homologues in bacterial genomes inspired us to investigate overall phylogeny. Here we present a comprehensive phylogenetic analysis of the Hip kinases in bacteria and archaea that expands on this diversity by revealing seven novel kinase families. Kinases of one family, encoded by monocistronic operons, consist of an N-terminal core kinase domain, a HipS-like domain and a HIRAN (HIP116 Rad5p N-terminal) domain. HIRAN domains bind single or double-stranded DNA ends. Moreover, five types of bicistronic kinase operons encode putative antitoxins with HipS-HIRAN, HipS, γδ-resolvase or Stl repressor-like domains. Finally, our analysis indicates that reversion of hipBA gene-order happened independently several times during evolution.ImportanceBacterial multidrug tolerance and persistence are problems of increasing scientific and medical significance. The first gene discovered to confer persistence was hipA, encoding the kinase toxin of the hipBA toxin-antitoxin (TA) module of E. coli. HipA-homologous kinases phosphorylate and thereby inactivate specific tRNA synthetases, thus inhibiting protein translation and cell proliferation. Here, we present a comprehensive phylogenetic analysis of bacterial Hip kinases and discover seven new families with novel operon structures and domains. Overall, Hip kinases are encoded by TA modules with at least 10 different genetic organizations, seven of which have not been described before. These results open up exciting avenues for the experimental analysis of the superfamily of Hip kinases.

Nucleolin promotes IRES-driven translation of foot-and-mouth disease virus by supporting the assembly of translation initiation complexes

Nucleolin (NCL), a stress-responsive RNA-binding protein, has been implicated in the translation of internal ribosome entry site (IRES)-containing mRNAs, which encode proteins involved in cell proliferation, carcinogenesis, and viral infection (Type I IRESs). However, the details of the mechanisms by which NCL participates in IRES-driven translation have not hitherto been described. Here, we identified NCL as a protein that interacts with the IRES of foot-and-mouth disease virus (FMDV), which is a Type II IRES. We also mapped the interactive regions within FMDV IRES and NCL in vitro. We found that NCL serves as a substantial regulator of FMDV IRES-driven translation but not of bulk cellular or vesicular stomatitis virus cap-dependent translation. NCL also modulates the translation of and infection by Seneca Valley virus (Type III-like IRES) and classical swine fever virus (Type III IRES), which suggests that its function is conserved in unrelated IRES-containing viruses. We also show that NCL affects viral replication by directly regulating the production of viral proteins and indirectly regulating FMDV RNA synthesis. Importantly, we observed that the cytoplasmic relocalization of NCL during FMDV infection is a substantial step for viral IRES-driven translation, and that NCL specifically promotes the initiation phase of the translation process by recruiting translation initiation complexes to viral IRES. Finally, the functional importance of NCL in FMDV pathogenicity was confirmed in vivo. Taken together, our findings demonstrate a specific function for NCL in selective mRNA translation, and identify a target for the development of a broad-spectrum class of antiviral interventions. IMPORTANCE FMDV usurps the cellular translation machinery to initiate viral protein synthesis via a mechanism driven by IRES elements. It allows the virus to shut down bulk cellular translation, while providing an advantage for its own gene expression. With limited coding capacity in its own genome, FMDV has evolved a mechanism to hijack host proteins to promote the recruitment of the host translation machinery, a process that is still not well understood. Here, we identified nucleolin (NCL) as a positive regulator of the IRES-driven translation of FMDV. Our study supports a model in which NCL relocalizes from the nucleus to the cytoplasm during the course of FMDV infection, where the cytoplasmic NCL promotes FMDV IRES-driven translation by bridging the translation initiation complexes with viral IRES. Our study demonstrates a previously uncharacterized role of NCL in the translation initiation of IRES-containing viruses, with important implications for the development of broad antiviral interventions.

Duck Hepatitis A Virus Type 1 Induces eIF2α Phosphorylation-Dependent Cellular Translation Shutoff via PERK/GCN2

Duck hepatitis A virus type 1 (DHAV-1) is one of the most deadly pathogens that endanger the duck industry. Most viruses usually turn off host translation after infection to facilitate viral replication and translation. For the first time report to our knowledge, DHAV-1 can induce eIF2α phosphorylation and inhibit cellular translation in duck embryo fibroblasts (DEFs). Moreover, the activity of DHAV-1 in the cells caused obvious eIF2α phosphorylation, which has nothing to do with the viral protein. Subsequently, we screened two kinases (PERK and GCN2) that affect eIF2α phosphorylation through inhibitors and shRNA. Notably, the role of GCN2 in other picornaviruses has not been reported. In addition, when the phosphorylation of eIF2α induced by DHAV-1 is inhibited, the translation efficiency of DEFs restores to a normal level, indicating that DHAV-1 induced cellular translation shutoff is dependent on eIF2α phosphorylation.

The viral protein NSP1 acts as a ribosome gatekeeper for shutting down host translation and fostering SARS-CoV-2 translation

SARS-CoV-2 coronavirus is responsible for Covid-19 pandemic. In the early phase of infection, the single-strand positive RNA genome is translated into non-structural proteins (NSP). One of the first proteins produced during viral infection, NSP1, binds to the host ribosome and blocks the mRNA entry channel. This triggers translation inhibition of cellular translation. In spite of the presence of NSP1 on the ribosome, viral translation proceeds however. The molecular mechanism of the so-called viral evasion to NSP1 inhibition remains elusive. Here, we confirm that viral translation is maintained in the presence of NSP1. The evasion to NSP1-inhibition is mediated by the cis-acting RNA hairpin SL1 in the 5’UTR of SARS-CoV-2. NSP1-evasion can be transferred on a reporter transcript by SL1 transplantation. The apical part of SL1 is only required for viral translation. We show that NSP1 remains bound on the ribosome during viral translation. We suggest that the interaction between NSP1 and SL1 frees the mRNA accommodation channel while maintaining NSP1 bound to the ribosome. Thus, NSP1 acts as a ribosome gatekeeper, shutting down host translation or fostering SARS-CoV-2 translation depending on the presence of the SL1 5’UTR hairpin. SL1 is also present and necessary for translation of sub-genomic RNAs in the late phase of the infectious program. Consequently, therapeutic strategies targeting SL1 should affect viral translation at early and late stages of infection. Therefore, SL1 might be seen as a genuine ‘Achille heel’ of the virus.

Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins

The ribosomal stalk proteins, RPLP1 and RPLP2 (RPLP1/2), which form the ancient ribosomal stalk, were discovered decades ago but their functions remain mysterious. We had previously shown that RPLP1/2 are exquisitely required for replication of dengue virus (DENV) and other mosquito-borne flaviviruses. Here, we show that RPLP1/2 function to relieve ribosome pausing within the DENV envelope coding sequence, leading to enhanced protein stability. We evaluated viral and cellular translation in RPLP1/2-depleted cells using ribosome profiling and found that ribosomes pause in the sequence coding for the N-terminus of the envelope protein, immediately downstream of sequences encoding two adjacent transmembrane domains (TMDs). We also find that RPLP1/2 depletion impacts a ribosome density for a small subset of cellular mRNAs. Importantly, the polarity of ribosomes on mRNAs encoding multiple TMDs was disproportionately affected by RPLP1/2 knockdown, implying a role for RPLP1/2 in multi-pass transmembrane protein biogenesis. These analyses of viral and host RNAs converge to implicate RPLP1/2 as functionally important for ribosomes to elongate through ORFs encoding multiple TMDs. We suggest that the effect of RPLP1/2 at TMD associated pauses is mediated by improving the efficiency of co-translational folding and subsequent protein stability.