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Methods comparison for ribosome profiling

ABSTRACT: Assessment of the effecacy of direction extraction of ribosome-protected mRNA fragments for ribosome profiling Immortalized human lymphocytes were analyzed by ribosome profiling protocols with or without ribosome purification

ORGANISM(S): Homo sapiens

SUBMITTER: David Reid

PROVIDER: E-GEOD-68008 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Methods comparison for ribosome profiling

Project description:Assessment of the effecacy of direction extraction of ribosome-protected mRNA fragments for ribosome profiling

2015-07-01 | GSE68008 | GEO

Widespread Co-translational RNA Decay Reveals Ribosome Dynamics

Project description:It is generally assumed that mRNAs undergoing translation are protected from decay. Here, we show that mRNAs are, in fact, co-translationally degraded. This is a widespread and conserved process affecting most genes, where 5′–3′ transcript degradation follows the last translating ribosome, producing an in vivo ribosomal footprint. By sequencing the ends of 5′ phosphorylated mRNA degradation intermediates, we obtain a genome-wide drug-free measurement of ribosome dynamics. We identify general translation termination pauses in both normal and stress conditions. In addition, we describe novel codon-specific ribosomal pausing sites in response to oxidative stress that are dependent on the RNase Rny1. Our approach is simple and straightforward and does not require the use of translational inhibitors or in vitro RNA footprinting that can alter ribosome protection patterns. RNA samples were quantified according to their 5’modification. Samples were quantified using 2 alternative methods. 5PSeq (measures 5’P RNAs) and 5PSeq-fragmented (measures the sequencing bias present in any sample by randomly fragmenting RNA, re-phosphorilating the 5’ends and subjecting the sample to 5PSeq; it is used as a negative control). Different strains of S. cerevisiae and S. pombe samples were analyzed. Cells were grown both in rich media (YPD or YES) and synthetic defined media (SD). Cells were subjected to different treatments such as inhibition of translation elongation with cyclohexamide (CHX), oxidative stress (0.2 mM H2O2) or inhibition of histidine biosyntesys (3-AT; 100mM 3-amino-1,2,4-triazole). S. cerevisiae samples grown in rich media were also analyzed after CHX treatment and sucrose fractionation into monosome and polyribosome fractionation. All samples were analyzed in biological duplicates.

2015-06-04 | E-GEOD-63120 | biostudies-arrayexpress

Ribosome profiling reveals features of normal and disease-associated mitochondrial translation

Project description:Ribosome profiling and RNAseq data on human BJ fibroblasts and cybrid cells using an adapted ribosome profiling protocol to improve detection of mitochondrial ribosome protected fragments 51-base length single read ribosome profiling data on human fibroblasts and cybrid cells using an adapted ribosome profiling protocol; and 51-base length single read RNAseq on polyA enriched RNA

2013-10-17 | E-GEOD-48933 | biostudies-arrayexpress

Ribosome hibernation factor promotes Staphyloccocal survival and differentially represses translation

Project description:We sequenced a total of 16 cDNA libararies derived from fragmented total mRNA and ribosome protected mRNAs from S. aureus hpf mutant (NE838) and its parental strain JE2 grown in tryptic soy broth (TSB) or minimal medium (MM). The data represented 2 independent biological replicates. Examination of the impact of hpf on global S.aureus translatome, mRNA abundance and the ribosome density along the transcripts.

2016-03-29 | E-GEOD-74197 | biostudies-arrayexpress

RIBO-seq analysis of YTHDF2-deficient pre-leukemic cells and control pre-leukemic cells

Project description:YTHDF2 is overexpressed in a broad spectrum of human acute myeloid leukemias (AML). To study the role of YTHDF2 on translation regulation in leukemia, c-Kit+ cells from foetal livers of Ythdf2fl/fl; Vav-iCre (Ythdf2CKO) and Ythdf2fl/fl (Ythdf2CTL) 14.5 dpc embryos were transduced with Meis1 and Hoxa9 oncogenes and serially re-plated to generate pre-leukemic cells. RIBO-seq libraries were then prepared from Ythdf2CKO (n=3) and Ythdf2CTL (n=3) pre-leukemic cells.

2019-04-05 | E-MTAB-7785 | biostudies-arrayexpress

Clarifying the translational pausing landscape in bacteria by ribosome profiling

Project description:The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with ribosome profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by ribosome profiling. Ribosome profiling (three replicates) and RNAseq (two replicates) of E. coli MG1655

2016-01-13 | E-GEOD-72899 | biostudies-arrayexpress

Ribosome profiling reveals features of normal and disease-associated mitochondrial translation

2013-10-17 | GSE48933 | GEO

Ribosome profiling of the genome-reduced bacterium Mycoplasma pneumoniae

Project description:In order to study quantitatively the translation efficiency in the genome-reduced bacterium Mycoplasma pneumoniae, we performed ribosome profiling (ribo-seq) in standard growth conditions. We first tested whether the ribosome profiling method could be applied to M. pneumoniae and assessed the quality of the obtained data. Mpn present some characteristics that differ from other model bacterial species where ribosome profiling has been established, like the absence of a cell wall, different lipid composition and low number of ribosomes (200-300 per cell compared with ~55,000 in E. coli). Thus, several aspects of the original method (Ingolia et al., 2009) had to be adapted. As a reference, we also performed ribosome profiling in E. coli. We also calibrated the ribosome P-site shift from the 3’ end of ribosome protected footprints (RPFs) by analyzing the metagene profile of a sample treated with tetracycline.

2023-07-01 | E-MTAB-11935 | biostudies-arrayexpress

A spectral analysis approach to detects actively translated open reading frames in high resolution ribosome profiling data

Project description:RNA sequencing protocols allow for quantifying gene expression regulation at each individual step, from transcription to protein synthesis. Ribosome Profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. Despite its great potential, a rigorous statistical approach to identify translated regions from Ribo-seq data is not yet available. To fill this gap, we developed RiboTaper, which quantifies the significance of the three-nucleotide periodicity of Ribo-seq reads via spectral analysis methods. Examination of RNA fragments protected by ribosome

2015-11-12 | E-GEOD-73136 | biostudies-arrayexpress

Mechanism of chaperone coordination during cotranslational protein folding in bacteria

Project description:Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally-distinct classes of chaperones promote de novo folding, suggesting that their activity is coordinated at the ribosome. Here we use biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We find that chaperone binding is disfavoured close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor subsequently recognises compact folding intermediates exposing extensive non-native surface and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates, and recruits DnaK to solvent-accessible sites in a sequence non-specific manner. Neither Trigger factor, DnaJ nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to create a protected space for protein maturation that extends well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.

2024-07-03 | PXD048638 | Pride

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