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This work employed pleuromutilin-assisted ribosome profiling using retapamulin (Ribo-RET) to identify genome-wide translation start sites in the human pathogen Streptococcus pneumoniae . We identified 114 unannotated intergenic small open reading frames (sORFs). Streptococcus pneumoniae , an opportunistic human pathogen, is the leading cause of community-acquired pneumonia and an agent of otitis media, septicemia, and meningitis. Although genomic and transcriptomic studies of S. pneumoniae have provided detailed perspectives on gene content and expression programs, they have lacked information pertaining to the translational landscape, particularly at a resolution that identifies commonly overlooked small open reading frames (sORFs), whose importance is increasingly realized in metabolism, regulation, and virulence. To identify protein-coding sORFs in S. pneumoniae , antibiotic-enhanced ribosome profiling was conducted. Using translation inhibitors, 114 novel sORFs were detected, and the expression of a subset of them was experimentally validated. Two loci associated with virulence and quorum sensing were examined in deeper detail. One such sORF, rio3 , overlaps with the noncoding RNA srf-02 that was previously implicated in pathogenesis. Targeted mutagenesis parsing rio3 from srf-02 revealed that rio3 is responsible for the fitness defect seen in a murine nasopharyngeal colonization model. Additionally, two novel sORFs located adjacent to the quorum sensing receptor rgg1518 were found to impact regulatory activity. Our findings emphasize the importance of sORFs present in the genomes of pathogenic bacteria and underscore the utility of ribosome profiling for identifying the bacterial translatome. IMPORTANCE This work employed pleuromutilin-assisted ribosome profiling using retapamulin (Ribo-RET) to identify genome-wide translation start sites in the human pathogen Streptococcus pneumoniae . We identified 114 unannotated intergenic small open reading frames (sORFs). The described procedures and data sets provide a model for microbiologists seeking to explore the translational landscape of bacteria. The biological roles of four sORF examples are characterized: two control the regulation of a cell-cell communication (quorum sensing) system, one contributes to the ability of S. pneumoniae to colonize the upper respiratory tract of mice, and a fourth governs the translation of PrfB, a protein enabling ribosome release at stop codons. We propose that Ribo-RET is a valuable approach to identifying unstudied microproteins and difficult-to-find pheromone genes used by Gram-positive organisms, whose genomes are replete with pheromone receptors.

Tomato ( Solanum lycopersicum L.), one of the most widely grown vegetable crops in the world, faces cracking problems before and after harvest. Fruit cracking reduces the commercial value and seriously affects the economic performance of the fruits by affecting the appearance and quality of the fruit. Clarifying the molecular mechanism underlying tomato fruit cracking is of great importance for selecting and breeding cracking-resistant varieties. At present, research on the molecular mechanism of tomato fruit cracking has made progress, but few studies have been conducted to explore the genes related to fruit cracking regulation using combined multi-omics analysis. We applied Ribo-seq (ribosome analysis sequencing) and RNA-seq (RNA-sequencing) techniques to uncover potential fruit cracking regulatory genes and improve the regulatory network of fruit cracking using extremely cracking-resistant (CR) and cracking-susceptible (CS) tomato genotypes. Combining these two sets of histological data and translation efficiency, 41 genes were identified to be associated with fruit cracking. The genes played functions on hormone synthesis (e.g. Solyc09g089580.4 , Solyc07g049530.3 ), reactive oxygen species regulation (e.g. Solyc08g080940.3 ), cell wall metabolism (e.g. Solyc04g071070.2 , Solyc03g123630.4 ), aquaporins activity (e.g. Solyc03g096290.3 , Solyc10g083880.2 ), cuticle and wax composition, as well as mineral elements transport (e.g. Solyc10g006660.3 , Solyc01g057770.3 ), while 10 of them were transcription factors (TF) (e.g. Solyc05g015850.4 , Solyc08g078190.2 ). Based on the investigation of the interaction relationship between these genes, the synergistic regulation of multi-gene tomato fruit cracking was predicted. This study suggests that the synergistic action of transcription and translation is an important molecular mechanism in regulating tomato fruit cracking. Highlights We report a comprehensive analysis of tomato cracking based on the RNA-seq (RNA-sequencing) and Ribo-seq (ribosome profiling sequencing) using extremely cracking-resistant and -susceptible tomatoes. The 41 possible genes regulating tomato fruit cracking were identified. Two regulation networks, including ethylene-cell wall metabolic pathway and transcription factor-water channel-mineral element were found to be involved in regulating tomato fruit cracking.

Protein synthesis is strictly regulated during replicative aging in yeast, but global translational regulation during replicative aging is poorly characterized. To conduct ribosome profiling during replicative aging, we collected a large number of dividing aged cells using a miniature chemostat aging device. Translational efficiency, defined as the number of ribosome footprints normalized to transcript abundance, was compared between young and aged cells for each gene. We identified more than 700 genes with changes greater than twofold during replicative aging. Increased translational efficiency was observed in genes involved in DNA repair and chromosome organization. Decreased translational efficiency was observed in genes encoding ribosome components, transposon Ty1 and Ty2 genes, transcription factor HAC1 gene associated with the unfolded protein response, genes involved in cell wall synthesis and assembly, and ammonium permease genes. Our results provide a global view of translational regulation during replicative aging, in which the pathways involved in various cell functions are translationally regulated and cause diverse phenotypic changes.

Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from Mycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of M. smegmatis in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.

Deep transcriptome sequencing has revealed the existence of many transcripts that lack long or conserved open reading frames (ORFs) and which have been termed long non-coding RNAs (lncRNAs). The vast majority of lncRNAs are lineage-specific and do not yet have a known function. In this study, we test the hypothesis that they may act as a repository for the synthesis of new peptides. We find that a large fraction of the lncRNAs expressed in cells from six different species is associated with ribosomes. The patterns of ribosome protection are consistent with the translation of short peptides. lncRNAs show similar coding potential and sequence constraints than evolutionary young protein coding sequences, indicating that they play an important role in de novo protein evolution. Despite the terms being largely interchangeable in modern language, ‘DNA’ and ‘gene’ do not mean the same thing. A gene is made of DNA and contains the instructions to make a protein, and it is the protein that performs the function of the gene. However, cells in the body also contain DNA that does not form genes. Far from being ‘junk’ DNA with no biological purpose; this DNA has a variety of roles, including affecting how other genes are used. To produce a protein, the DNA sequence of a gene is transcribed into an intermediate molecule called RNA, which is then translated to produce a protein. So-called long non-coding RNA (lncRNA) molecules are also transcribed from DNA, but whether these are translated to make proteins has been a subject of much debate. Indeed, the function of the vast majority of lncRNA molecules is unknown. Ruiz-Orera et al. analyzed RNA sequences collected from earlier experiments on six different species—humans, mice, fish, flies, yeast, and a plant—and found nearly 2500 as yet unstudied lncRNAs in addition to those previously identified. Many of the lncRNAs that Ruiz-Orera et al. investigated could be found lodged inside the cellular machinery used to translate RNA into proteins. Furthermore, these lncRNA molecules are oriented in the machinery as if they are primed and ready for translation, suggesting that many lncRNAs do produce proteins. However, it is unclear how many of these proteins have a useful function. Very few lncRNAs were found in more than one species, suggesting that they have evolved recently. The properties of lncRNA molecules also show many similarities with the properties of ‘young’—recently evolved—genes that are known to produce proteins. The combined findings of Ruiz-Orera et al. therefore suggest that lncRNAs are important for developing new proteins. The emergence of proteins with new functions has been an important driving force in evolution, and this work provides important clues into the first steps of this process.

Translating ribosomes slow down or completely stall when they encounter obstacles on mRNAs. Such events can lead to ribosomes colliding with each other and forming complexes of two (disome), three (trisome) or more ribosomes. While these events can activate surveillance pathways, it has been unclear if collisions are common on endogenous mRNAs and whether they are usually detected by these cellular pathways. Recent genome-wide surveys of collisions revealed widespread distribution of disomes and trisomes across endogenous mRNAs in eukaryotic cells. Several studies further hinted that the recognition of collisions and response to them by multiple surveillance pathways depend on the context and duration of the ribosome stalling. This review considers recent efforts in the identification of endogenous ribosome collisions and cellular pathways dedicated to sense their severity. We further discuss the potential role of collided ribosomes in modulating co-translational events and contributing to cellular homeostasis.

Protein synthesis represents a major metabolic activity of the cell. However, how it is affected by aging and how this in turn impacts cell function remains largely unexplored. To address this question, herein we characterized age-related changes in both the transcriptome and translatome of mouse tissues over the entire life span. We showed that the transcriptome changes govern those in the translatome and are associated with altered expression of genes involved in inflammation, extracellular matrix, and lipid metabolism. We also identified genes that may serve as candidate biomarkers of aging. At the translational level, we uncovered sustained down-regulation of a set of 5′-terminal oligopyrimidine (5′-TOP) transcripts encoding protein synthesis and ribosome biogenesis machinery and regulated by the mTOR pathway. For many of them, ribosome occupancy dropped twofold or even more. Moreover, with age, ribosome coverage gradually decreased in the vicinity of start codons and increased near stop codons, revealing complex age-related changes in the translation process. Taken together, our results reveal systematic and multidimensional deregulation of protein synthesis, showing how this major cellular process declines with age.

Translation regulation is critical for early mammalian embryonic development 1 . However, previous studies had been restricted to bulk measurements 2 , precluding precise determination of translation regulation including allele-specific analyses. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key mechanism regulating genes involved in centrosome organization and N 6 -methyladenosine modification of RNAs. Our high-coverage measurements enabled, to our knowledge, the first analysis of allele-specific ribosome engagement in early development. These led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allele-biased expression. By integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. The Ribo-ITP approach will enable numerous applications by providing high-coverage and high-resolution ribosome occupancy measurements from ultra-low input samples including single cells. A single-cell ribosome profiling method can provide data at the level of allele-specific ribosome engagement in early development.

Stop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418-induced miscoding alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1). Many genes provide a set of instructions needed to build a protein, which are read by structures called ribosomes through a process called translation. The genetic information contains a short, coded instruction called a stop codon which marks the end of the protein. When a ribosome finds a stop codon it should stop building and release the protein it has made. Ribosomes do not always stop at stop codons. Certain chemicals can actually prevent ribosomes from detecting stop codons correctly, and aminoglycosides are drugs that have exactly this effect. Aminoglycosides can be used as antibiotics at low doses because they interfere with ribosomes in bacteria, but at higher doses they can also prevent ribosomes from detecting stop codons in human cells. When ribosomes do not stop at a stop codon this is called readthrough. There are different types of stop codons and some are naturally more effective at stopping ribosomes than others. Wangen and Green have now examined the effect of an aminoglycoside called G418 on ribosomes in human cells grown in the laboratory. The results showed how ribosomes interacted with genetic information and revealed that certain stop codons are more affected by G418 than others. The stop codon and other genetic sequences around it affect the likelihood of readthrough. Wangen and Green also showed that sequences that encourage translation to stop are more common in the area around stop codons. These findings highlight an evolutionary pressure driving more genes to develop strong stop codons that resist readthrough. Despite this, some are still more affected by drugs like G418 than others. Some genetic conditions, like cystic fibrosis, result from incorrect stop codons in genes. Drugs that promote readthrough specifically in these genes could be useful new treatments.

Emerging data have shown that previously defined noncoding genomes might encode peptides that bind human leukocyte antigen (HLA) as cryptic antigens to stimulate adaptive immunity 1 , 2 . However, the significance and mechanisms of action of cryptic antigens in anti-tumour immunity remain unclear. Here mass spectrometry of the HLA class I (HLA-I) peptidome coupled with ribosome sequencing of human breast cancer samples identified HLA-I-binding cryptic antigenic peptides that were noncanonically translated by a tumour-specific circular RNA (circRNA): circFAM53B. The cryptic peptides efficiently primed naive CD4 + and CD8 + T cells in an antigen-specific manner and induced anti-tumour immunity. Clinically, the expression of circFAM53B and its encoded peptides was associated with substantial infiltration of antigen-specific CD8 + T cells and better survival in patients with breast cancer and patients with melanoma. Mechanistically, circFAM53B-encoded peptides had strong binding affinity to both HLA-I and HLA-II molecules. In vivo, administration of vaccines consisting of tumour-specific circRNA or its encoded peptides in mice bearing breast cancer tumours or melanoma induced enhanced infiltration of tumour-antigen-specific cytotoxic T cells, which led to effective tumour control. Overall, our findings reveal that noncanonical translation of circRNAs can drive efficient anti-tumour immunity, which suggests that vaccination exploiting tumour-specific circRNAs may serve as an immunotherapeutic strategy against malignant tumours. The tumour-specific circular RNA FAM53B is highly immunogenic and can induce anti-tumour responses in mouse models of breast cancer and melanoma, expanding the repertoire of anticancer targets for development.