Similar Datasets
Project description:Evaluating levels of Vaccinia viral mRNA translation
| PRJNA952251 | ENA
Project description:Many viruses potently inhibit host protein synthesis while employing unconventional strategies to sustain their own translation, but how and why certain cellular mRNAs continue to be translated remains unclear. Here, we show that during shutoff by Vaccinia Virus (VacV) several host mRNAs increase in polysome occupancy but few, topped by JUN, result in increased protein abundance. Translation of viral mRNAs depended on the small ribosomal protein, Receptor for Activated C Kinase 1 (RACK1) and the eukaryotic Initiation Factor, eIF3. Cryo-electron microscopy (cryo-EM) further showed that eIF3 bound to virus-modified RACK1-containing 40S subunits that exhibit altered head rotation during infection. By contrast, RACK1 and eIF3 were dispensable for JUN translation. Moreover, structurally distinct 5’ untranslated regions (5’UTRs) in viral versus JUN mRNAs conferred differential eIF3 dependencies. Altogether, we reveal how distinct modes of non-canonical initiation support the production of both host and viral proteins that facilitate poxvirus replication despite infection-induced shutoff.
2024-10-30 | GSE278320 | GEO
Project description:Regulation of the efficiency with which an mRNA is translated into proteins represents a key mechanism for controlling gene expression. Such regulation impacts the number of actively translating ribosomes per mRNA molecule, referred to as translation efficiency (TE), which can be monitored using ribosome profiling and RNA-seq, or by evaluating the position of an mRNA in a polysome gradient. Here we show that in budding yeast, under nutrient limiting conditions, the commonly used translation inhibitor cycloheximide induces rapid transcriptional upregulation of hundreds of genes involved in ribosome biogenesis. Cycloheximide also prevents translation of these newly transcribed messages, leading to an apparent drop in TE of these genes under conditions that include key transitions of the yeast metabolic cycle, meiosis, and amino acid starvation; an effect which is abolished when cycloheximide pretreatment is omitted. This response requires TORC1 signaling, and is modulated by the genetic background as well as the vehicle used to deliver the drug. The present work highlights an important caveat to the use of translation inhibitors when measuring TE, and will hopefully aid in future experimental design as well as interpretation of prior results
2019-03-08 | GSE125038 | GEO
Project description:We report the application of polysome profiling sequencing technology for high-throughput transcriptomics and translatomics in mammalian cells. We compare reduction of Dap5 to control in metastatic breast cancer cells 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 factor and receptor capped mRNAs and their mRNA targets involved in cell survival, motility, DNA repair and translation initiation, among other mRNAs.
2018-06-30 | GSE115142 | GEO
Project description:Cells infected by influenza virus mount a large-scale antiviral response and most cells ultimately initiate cell-death pathways in an attempt to suppress viral replication. We performed a CRISPR/Cas9-knockout selection designed to identify host factors required for replication following viral entry. We identified a large class of presumptive antiviral factors that unexpectedly act as important pro-viral enhancers during influenza virus infection. One of these, IFIT2, is an interferon-stimulated gene with well-established antiviral activity but limited mechanistic understanding. As opposed to suppressing infection, we show here that IFIT2 is instead repurposed by influenza virus to promote viral gene expression. CLIP‐seq demonstrated that IFIT2 binds directly to viral and cellular mRNAs in AU‐rich regions, with bound cellular transcripts enriched in interferon‐stimulated mRNAs. Polysome and ribosome profiling revealed that IFIT2 prevents ribosome pausing on bound mRNAs. Together, the data link IFIT2 binding to enhanced translational efficiency for viral and cellular mRNAs and ultimately viral replication. Our findings establish a model for the normal function of IFIT2 as a protein that increases translation of cellular mRNAs to support antiviral responses and explain how influenza virus uses this same activity to redirect a classically antiviral protein into a pro-viral effector.
2022-02-03 | GSE196010 | GEO
Project description:Upon detection of viral infections, cells activate the expression of type I interferons (IFNs) and pro-inflammatory cytokines to control viral dissemination. As part of their antiviral response, cells also trigger the translational shutoff response which prevents translation of viral mRNAs and cellular mRNAs in a non-selective manner. Intriguingly, mRNAs encoding for antiviral factors bypass this translational shutoff, suggesting the presence of additional regulatory mechanisms enabling expression of the self-defence genes. Here, we identified the dsRNA binding protein ILF3 as an essential host factor required for efficient translation of the central antiviral cytokine, IFNB1, and a subset of interferon-stimulated genes. By combining polysome profiling and next-generation sequencing, ILF3 was also found to be necessary to establish the dsRNA-induced transcriptional and translational programs. We propose a central role for the host factor ILF3 in enhancing expression of the antiviral defence mRNAs in cellular conditions where cap-dependent translation is compromised. In this dataset we use polysome profiling in combination with RNA-seq to investigate the effect of ILF3 on the translation of mRNAs in HeLa cells in homeostasis and the antiviral response.
2019-10-25 | GSE130742 | GEO
Project description:We have generated stable human ESCs (H9) expressing control or DAP5-targeting shRNA. Polysome profiles reveal no major changes in overall translation. PolyA+ RNA and RNA accociated with heavy polysomal fractions were purified in biological duplicates and sequenced using Illumina HiSeq 2000 instrument. We identified 122 potential mRNA targets of DAP5 translation that display reduced ribosomal loading, and hence reduced translation, in the absence of DAP5.
2016-09-14 | GSE80467 | GEO
Project description:Transcript leaders (TLs) can have profound effects on mRNA translation and stability. To map TL boundaries genome-wide, we developed TL-Sequencing (TL-Seq), a technique combining enzymatic capture of m7G-capped mRNA 5'-ends with high-throughput sequencing. TL-Seq identified mRNA start sites for the majority of yeast genes and revealed many examples of intragenic TL heterogeneity. Transcription initiation sites occur in at least 5% of protein-coding regions and are concentrated near the 5'ends of ORFs. These truncated mRNAs are translated, based on ribosome density analysis. Translation Associated TL-Seq (TATL-Seq), which combines TL-Seq with polysome fractionation, revealed substantial differences in translation of alternative TL isoforms. Globally, while some TL features are associated with poor translation and nonsense-mediated mRNA decay (uAUGs, very short length), others (secondary structure, long length) have much less impact than predicted from analyses of individual genes. TL-Seq and TATL-Seq can be applied to any eukaryote to investigate TL-mediated regulation of gene expression. TL-Seq (+/- pyrophosphatase), TATL-Seq (TL-Seq libraries from polysome gradient)
2013-04-03 | E-GEOD-39074 | biostudies-arrayexpress
Project description:How viruses, such as the emerging mosquito-borne Chikungunya virus (CHIKV), express their genomes at high levels despite an enrichment in suboptimal codons remains a puzzling question. By integrating subcellular fractionation and transcriptome-wide analyses of translation in CHIKV-infected human cells, we demonstrate an unanticipated virus-induced reprogramming of the host translation machinery to favor translation of viral RNA over cellular genes featuring optimal codon usage. This reprogramming was specifically apparent at the endoplasmic reticulum (ER), the preferred translation compartment of CHIKV RNA, and it is mediated by the wobble uridine 34 tRNA modification enzyme KIAA1456 whose expression is enhanced upon viral infection. Since KIAA1456 itself is encoded by a CHIKV-like codon usage, infection triggers a positive feed-back loop that ensures efficient virus protein production. Our findings demonstrate an unprecedented interplay of viruses with the host tRNA epitranscriptome to favor viral protein expression.
2022-06-17 | GSE143390 | GEO
Project description:Translation is the process by which genetic information from mRNA is decoded to produce proteins. Since mRNA levels alone do not predict protein amounts accurately, understanding translational control is essential. Ribosome profiling revealed that translation initiation is the main rate-limiting step, with rates varying up to 100-fold across mRNAs, while elongation rates vary less (~20-fold). Furthermore, only a small fraction of the variation in translation rates is explained by known determinants, indicating that much remains to be understood in the prediction of protein outputs of individual mRNAs. Current machine learning models would benefit from more data on endogenous mRNAs. To address this, here we report steady-state and dynamic multi-omics data from human liver cancer cell lines, specifically i) ribosome profiling of unperturbed cells and following translation initiation block to trace elongation (run-off ribosome profiling), ii) protein synthesis rates estimated by pulsed stable isotope labeled amino acids in cell culture (pSILAC), and iii) mean ribosome load on individual mRNAs determined by mRNA sequencing of polysome fractions (polysome profiling). Predicting protein output from mRNAs is crucial for applications like protein expression engineering or mRNA vaccine designing.
2024-11-25 | MSV000096514 | MassIVE