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A distinct mechanism governs translation initiation and its regulation in Staphylococcus aureus [RNA-Seq]

ABSTRACT: Retapamulin-assisted ribosome profiling (Ribo-Ret) reveals translation initiation sites at a translatome-wide scale. Here we employed Ribo-Ret in S. aureus, utilizing either MNase or RNAse 1 for ribosomal protected footprint (RPF) generation, significanlty improving the obtainable resolution at translation start sites with the latter. The improved resolution helped to identify precise start codon selection, revealing distinct features of the S. aureus translation initiation mechanism as well as multiple unrecognized small open reading frames (sORFs). Furthermore, we identified novel regulators of the translation initiation step in this major human pathogen.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE299222 | GEO | 2025/12/31

REPOSITORIES: GEO

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A distinct mechanism governs translation initiation and its regulation in Staphylococcus aureus [Ribo-Seq]

Project description:Retapamulin-assisted ribosome profiling (Ribo-Ret) reveals translation initiation sites at a translatome-wide scale. Here we employed Ribo-Ret in S. aureus, utilizing either MNase or RNAse 1 for ribosomal protected footprint (RPF) generation, significanlty improving the obtainable resolution at translation start sites with the latter. The improved resolution helped to identify precise start codon selection, revealing distinct features of the S. aureus translation initiation mechanism as well as multiple unrecognized small open reading frames (sORFs). Furthermore, we identified novel regulators of the translation initiation step in this major human pathogen.

2025-12-31 | GSE299221 | GEO

Retapamulin-assisted Ribo-seq reveals the alternative bacterial proteome

Project description:Unorthodox rules of extracting genetic information enable proteome expansion without increasing the genome size. The use of alternative translation initiation sites achieves this goal by allowing production of more than one protein from a single gene. Although several such examples have been serendipitously found in bacteria, genome-wide experimental mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin treatment followed by Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the annotated Escherichia coli genes but, strikingly, it also revealed putative alternative internal start sites in a number of genes. Experimental evidence demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose translation is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the ‘alternative’ bacterial proteome. In addition to proteome expansion, the internal start sites may play regulatory role in gene expression.

2019-03-04 | GSE122129 | GEO

Exploring the hidden world of Methanosarcina mazei: Unravelling the secrets of its small proteome and its nitrogen-dependent regulation

Project description:The mesophilic methanogenic archaeal model organism Methanosarcina mazei strain Gö1, is crucial for climate and environmental research due to its ability to produce methane from H2 plus CO2, acetate, and methylamines. Here, we established the first Ribo-seq protocol for M. mazei strain Gö1 under two growth conditions (nitrogen sufficiency (+N) versus nitrogen limitation (-N)). Translation of 93 previously annotated and 311 novel small open reading frames (sORFs), coding for proteins ≤ 70 amino acids in length was predicted with high confidence based on Ribo-seq data. Epitope tagging followed by immunoblotting analysis confirmed the translation of 12 out of 15 selected novel sORFs, the remaining three were validated by LC-MS-MS analysis. In total, LC-MS-MS analysis validated translation for 60 annotated sORFs and 26 novel sORFs. A comprehensive differential expression analysis revealed that 29 of 311 novel sORFs were differentially regulated in response to nitrogen availability at the transcriptional and 49 at the translational level. Several reported sRNAs are now emerging as dual-functional sRNAs, including sRNA154, the central regulatory sRNA in regulation of nitrogen metabolism. Numerous of the novel sORFs identified in this study are conserved in Methanosarcina species, some of which showed a phenotype when overproduced, pinpointing important physiological functions. Overall, the comprehensive analysis opens a new avenue to start elucidating the function(s) of small proteins in M. mazei.

2024-08-02 | PXD045039 | Pride

Translational initiation in E. coli occurs at the correct sites genome-wide in the absence of mRNA-rRNA base-pairing

Project description:Shine-Dalgarno (SD) motifs are thought to play an important role in translational initiation in bacteria. Paradoxically, ribosome profiling studies in E. coli show no correlation between the strength of the SD in an mRNA and how efficiently it is translated. Performing profiling on ribosomes with altered anti-Shine-Dalgarno sequences, we reveal a genome-wide correlation between SD strength and ribosome occupancy that was previously masked by other contributing factors. Using the antibiotic retapamulin to trap initiation complexes at start codons, we find that the mutant ribosomes select start sites correctly, arguing that start sites are hard-wired for initiation through the action of other mRNA features. We show that A-rich sequences upstream of start codons promote initiation. Taken together, our genome-wide study reveals that SD motifs are not necessary for the ribosome to select where initiation occurs, though they do affect how efficiently initiation occurs at sites whose other features support initiation.

2020-02-21 | GSE135906 | GEO

Assembly and functionality of the ribosome with tethered subunits

Project description:Ribo-T is an engineered ribosome whose small and large subunits are tethered together by insertion of circularly permutated 23S rRNA into 16S rRNA [Orelle, C., Carlson, E. D., Szal, T., Florin, T., Jewett, M. C., Mankin, A.S. Protein synthesis by ribosomes with tethered subunits. Nature 524, 119-124 (2015)]. Whether the growth defects are related to problems with Ribo-T functionality is unclear. The translation in Ribo-T cells was examined by ribosome profiling (Ribo-seq) and RNA sequencing (RNA-seq). Besides the control cells carrying dissociable WT ribosome (SQ171), RIBO-T cells are the original SQ171 strain transformed with pRibo-T plasmid and cured of the plasmid carrying wt rRNA that was evolved to have improved growth characteristics. The Ribo-T strain carries two mutations: a nonsense mutation in the Leu22 codon of the ybeX gene and a missense mutation in codon 549 of the rpsA gene encoding ribosomal protein S1. Analysis of Ribo-T performance by ribosome profiling showed a notably higher ribosome occupancy of the start codons and somewhat increased occupancy at the stop codons of many genes, suggesting that subunit tethering specifically impairs the initiation and termination stages of translation.

2019-02-25 | GSE119454 | GEO

The small proteome of the nitrogen-fixing plant symbiont Sinorhizobium meliloti

Project description:Ribosome profiling was applied for the first time on S. meliloti to generate a translatome map. Our work reveals active translation for several ORFs including small ORFs (sORFs). In addition, our ribosome profiling data led to the discovery of several translated non-annotated sORFs. Translation of these non-annotated sORFs was validated by proteomics and western blotting.

2023-02-14 | GSE206492 | GEO

eIF4E/mTOR-independent mRNA translation plasticity conferred by DAP5/eIF3d is required for breast cancer cell mesenchymal transition and metastasis

Project description:We report the application of polysome profiling sequencing technology for high-throughput transcriptomics and translatomics in 4T1 cells. We compare reduction of Dap5 to control in a BALB/c mouse breast cancer cell line in transcription and polysome enriched translation using RNA sequencing. Genome-wide transcriptomic and translatomic analyses indicate that DAP5 is required for translation of many transcription factors and their mRNA targets involved in EMT, angiogenesis, DNA repair and translation initiation, among other mRNAs. Metastatic potential of cells is significanlty decreased upon Dap5 silencing as confirmed by different in vivo models.

2023-06-01 | GSE188733 | GEO

MHC-I peptidome analysis of human foreskin fibroblasts

Project description:Ribosome profiling (Ribo-seq) recently revealed the expression of thousands of short open reading frames (sORFs) in eukaryotic cells. They encode for a class of instable peptides, which evade experimental validation by whole-proteome mass spectrometry. Here, we show that computational elimination of experimental noise from Ribo-seq data unravels fundamental new aspects of sORF biology. Based on the revised annotation of cellular sORFs, we show that sORF-derived peptides are efficiently incorporated into MHC-I. sORFs thus encode a novel class of stress-responsive antigens in human cells. Our findings have broad implications on the functional, regulatory and immunogenic role of sORFs in fundamental cellular processes like infection and cancer.

2018-04-05 | PXD007203 | Pride

Synonymous codon usage regulates translation initiation

Project description:Ribo-seq with firefly luciferase optimality reporters to determine whether incomplete translation and/or inhibited translation initiation is responsible for the reduced ribosome occupancy on nonoptimal transcripts.

2022-05-16 | GSE202900 | GEO

Long-RNA sequencing and ribosome profiling of inflamed beta-cells reveal an extensive translatome landscape

Project description:Using a transcriptome-wide profiling approach to map translation initiation start sites in human beta-cells under standard and inflammatory conditions, we identifies non-canonical start sites and translation initiation within lncRNA.

2021-02-23 | GSE167223 | GEO

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