Project description:Ribosome profiling (Ribo-Seq) (maps positions of translating ribosomes on the transcriptome) analysis of human (RD) cells infected with enterovirus strains EV7, EV71, and PV1.
Project description:We present a genome-wide assessment of small open reading frames (smORF) translation by ribosomal profiling of polysomal fractions in Drosophila S2 cell. In this way, mRNAs bound by multiple ribosomes and hence actively translated can be isolated and distinguished from mRNAs bound by sporadic, putatively non-productive single ribosomes or ribosomal subunits. Ribosomal profiling of large and small polysomal fractions in Drosophila S2 cells to assess translation of smORFs
Project description:Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eIF2. However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a ~4.5-fold general translational repression, the protein coding ORFs of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated uORF that repress translation of the main coding ORF under normal conditions. Site specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely in SLC35A4 and MIEF1) encode functional protein products. Ribosome profiling of sodium arsenite treated cells for examination of translational response to induction of eIF2 phosphoylation
Project description:Murine 17Cl-1 cells were infected with the model Betacoronavirus mouse hepatitis virus (MHV) strain A59 and subjected to ribosome profiling to observe changes in the host translatome in infected cells compared to mock-infected cells. Samples were harvested at 5 and 8 hpi by flash-freezing, without cycloheximide pre-treatment. RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 25-35nt long. Fragments were cloned into adapters using the TruSeq small RNA adapter kit and sequenced on Illumina NextSeq, then differential translation efficiency analysis was carried out on the data deposited data. One uninfected sample was treated with 2ug/mL tunicamycin for 6 hours before harvesting as a positive control for unfolded protein response activation.
Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. Overall, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. Ribosome profiling of lactimidomycin and cycloheximide treated human Jurkat T-lymphocytes
Project description:Cell lines derived from Chlorocebus sabaeus kidney were infected with an isolate of PRRSV-1 or PRRSV-2 and ribosome profiling was performed (this entry) in parallel with RNASeq (see related accession number). These datasets were used to analyse the viral and host translatome, frameshifting on the viral genome, and putative frameshift-related ribosome pausing events. For the PRRSV-1 experiments, MA-104 cells were infected with an isolate based on the Porcilis vaccine strain (KJ127878.1 but with several accumulated mutations, see associated publication) and harvested at 8 hpi after pre-treatment with cycloheximide (CHX). For the PRRSV-2 experiments, MARC-145 cells were infected with SD95-21 PRRSV (KC469618.1), and a mutant thereof (KO2). One group of samples was harvested at 9 hpi after pre-treatment with CHX, and another group of samples was harvested at 3, 6, 9 and 12 hpi by flash-freezing without CHX pre-treatment. For all samples, RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 25-34 nt (PRRSV-1-infected samples, CHX-pre-treated PRRSV-2- or mock-infected samples, and non-CHX-pre-treated PRRSV-2- or mock-infected 9 hpi replicate one samples) or 19-34 nt long (all other samples). Fragments were cloned into adapters based on the TruSeq small RNA adapters. For all PRRSV-2-infected (or mock-infected) libraries, adapters with an additional seven random nucleotides at the 5′-end of the 3′-adapter and the 3′-end of the 5′-adapter were used. For PRRSV-1 replicate one, no random nucleotides were present on the adapters, and for PRRSV-1 replicate two, 14 random nucleotides were present at the 5′-end of the 3′-adapter. Libraries were sequenced on the Illumina NextSeq 500 platform as a single-end run. Non-CHX-pre-treated PRRSV-2-infected 9 hpi replicate two libraries were uploaded under a separate accession number due to differences in the size selection and sequencing protocol - see associated paired-end entry. Note that sample nomeclature (including replicate numbers) is consistent between this and the two related accessions, and RiboSeq libraries are matched with RNASeq libraries, which were prepared from the same lysate. The noCHX_Ribo_9hpi_mock_3 library is deliberately absent as this was a poor quality library.
Project description:MARC-145 cells were infected with porcine reproductive and respiratory syndrome virus (PRRSV)-2 isolate SD95-21 (KC469618.1), and a mutant thereof (KO2), and subjected to ribosome profiling to analyse the viral and host translatome, frameshifting on the viral genome, and putative frameshift-related ribosome pausing events. Samples were harvested at 9 hpi by flash-freezing, without cycloheximide pre-treatment. RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 19-80nt long. Fragments were cloned into adapters based on the TruSeq small RNA adapters, with an additional seven random nucleotides at the 5′-end of the 3′-adapter and the 3′-end of the 5′-adapter. Libraries were sequenced on the Illumina NextSeq 500 platform as a paired-end run. Note that sample nomeclature (including replicate numbers) is consistent between this and the two related accessions, and RiboSeq libraries are matched with RNASeq libraries, which were prepared from the same lysate.
Project description:BSR cells were infected with the Cardiovirus B archetype, Theiler’s murine encephalomyelitis virus (TMEV), and mutants thereof, and subjected to ribosome profiling to analyse ribosome collision events on the viral genome. Samples were harvested at 10 hpi by flash-freezing, without cycloheximide pre-treatment. RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 35-65nt long, in order to capture fragments protected by disomes. Fragments were cloned into adapters based on the TruSeq small RNA adapter kit, with an additional seven random nucleotides at 5′-end of the 3′-adapter. Libraries were sequenced on the Illumina NextSeq 500 platform.
Project description:BSR cells were infected with the Cardiovirus B archetype, Theiler’s murine encephalomyelitis virus (TMEV), and mutants thereof, and subjected to ribosome profiling to analyse the viral translatome, frameshifting, and ribosome pausing events. Samples were harvested at 10 hpi by flash-freezing, without cycloheximide pre-treatment. RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 19-35nt long. Fragments were cloned into adapters based on the TruSeq small RNA adapters, with an additional seven random nucleotides at the 5′-end of the 3′-adapter and the 3′-end of the 5′-adapter. Libraries were sequenced on the Illumina NextSeq 500 platform.
Project description:Ribosome profiling (RiboSeq) (maps positions of translating ribosomes on the transcriptome) and RNASeq (quantifies the transcriptome) analysis of murine L929 cells infected with encephalomyocarditis virus vMC0. Samples comprise 2, 4, 6 and 8 h post infection RiboSeq timecourses for WT virus and a shift mutant [SS] (8 samples), and RiboSeq and RNASeq at 8 h post infection for WT virus and 5 mutant viruses (shift site [SS], stem-loop [WT-SL], shift site and stem-loop [SS-SL], StopGo [LV], and StopGo, shift site and stem-loop [LV-SS-SL]) (12 samples).