Transcriptome-wide profiling of translation efficiency using mRNA-seq and Ribo-seq in ER stress-induced NIH3T3 cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE103667 | GEO | 2018/01/01
SECONDARY ACCESSION(S): PRJNA403993
REPOSITORIES: GEO
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Project description:We report systematical profiling of translation efficiency and mRNA stability dependent on the dynamics of poly(A)-tail length in stress conditions of human cells. In this study, we developed a new feasible method measuring poly(A)-tail length called TED-seq and applied it to investigate the change of mRNA's poly(A)-tail lengths in ER stress pharmacologically induced by thapsigargin (THAP). Combined with other global RNA analyses such as RNA-seq, Ribo-seq and PRO-seq, we observed that ER stress induced lenthening poly(A)-tail length, in particular of ER-stress-regulators, upon ER stress. More specifically, these mRNAs are translationally de-repressed and more stabilized based on increase in poly(A)-tail length. We also identified that insoluble fractions which include stress-induced RNA-granules have overall shorter length of poly(A) tail. Taken together, our data suggest that poly(A)-tail lengths are dynamically regulated and influence both translation efficiency and mRNA stability in ER stress.
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Project description:METTL16 is a member of methyltransferase like (METTL) family. Unlike well-studied METTL3 and METTL14, we found a much higher percentage of METTL16 is localized in the cytosol. The subcellular distribution holds the ability to potentiate translation efficiency. Via Far-western blotting and Co-Immunoprecipitation (Co-IP) assays, we have identified the direct interactions between METTL16 and eukaryotic initiation factor 3 (eIF3) a and b. Via cross-linking immunoprecipitation and qPCR (CLIP-qPCR), we have discovered the direct associations between METTL16 and rRNAs. The METTL16-eIF3a/b and METTL16-rRNAs interactions induce the binding between eIF3 and 18S rRNA, promote the formation of 43S preinitiation complex, and eventually expedite translation initiation, the rate-limiting step of translation. To determine the exact effects of METTL16 on translation efficiency, we performed ribosome profiling (Ribo-seq) with HEK293T upon CRISPR-Cas9-induced METTL16 knockout. To guarantee the repeatability and avoid any potential off-target effects, we included 3 distint sgRNAs against METTL16.
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Project description:Global analysis of translation regulation has recently been enabled by the development of Ribosome Profiling, or Ribo-seq, technology. This approach provides maps of ribosome activity for each expressed gene in a given biological sample. Measurements of translation efficiency are generated when Ribo-seq data is analyzed in combination with matched RNA-seq gene expression profiles. Existing computational methods for identifying genes with differential translation across samples are based on sound principles, but require users to choose between accuracy and speed.We present Riborex, a computational tool for mapping genome-wide differences in translation efficiency. Riborex shares a similar mathematical structure with existing methods, but has a simplified implementation. Riborex directly leverages established RNA-seq analysis frameworks for all parameter estimation, providing users with a choice among robust engines for these computations. The result is a method that is dramatically faster than available methods without sacrificing accuracy.https://github.com/smithlabcode/riborex.andrewds@usc.edu.Supplementary data are available at Bioinformatics online.
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Project description:Thousands of small Open Reading Frames (smORFs) with the potential to encode small peptides of fewer than 100 amino acids exist in our genomes. However, the number of smORFs actually translated, and their molecular and functional roles are still unclear. In this study, we present a genome-wide assessment of smORF translation by ribosomal profiling of polysomal fractions in Drosophila. We detect two types of smORFs bound by multiple ribosomes and thus undergoing productive translation. The 'longer' smORFs of around 80 amino acids resemble canonical proteins in translational metrics and conservation, and display a propensity to contain transmembrane motifs. The 'dwarf' smORFs are in general shorter (around 20 amino-acid long), are mostly found in 5'-UTRs and non-coding RNAs, are less well conserved, and have no bioinformatic indicators of peptide function. Our findings indicate that thousands of smORFs are translated in metazoan genomes, reinforcing the idea that smORFs are an abundant and fundamental genome component.
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Project description:Ribosome profiling (Ribo-Seq) is a highly sensitive method to quantify ribosome occupancies along individual mRNAs on a genome-wide scale. Hereby, ribosome-protected fragments (= footprints) are generated by nuclease digestion, isolated, and sequenced together with the corresponding randomly fragmented input samples, to determine ribosome densities (RD). For library preparation, equal amounts of total RNA are used. Subsequently, all transcript fragments are subjected to linker ligation, cDNA synthesis, and PCR amplification. Importantly, the number of reads obtained for every transcript in input and footprint samples during sequencing depends on sequencing depth and library size, as well as the relative abundance of the transcript in the sample. However, the information pertaining to the absolute amount of input and footprint sequences is lost during sample preparation, hence ruling out any conclusion whether translation is generally suppressed or activated in one condition over the other. Therefore, the RD fold-changes determined for individual genes do not reflect absolute regulation, but have to be interpreted as relative to bulk mRNA translation. Here, we modified the original ribosome profiling protocol that was first established by Ingolia et al. (2009), by adding small amounts of yeast lysate to the mammalian lysates of interest as a spike-in. This allows us to not only detect changes in the RD of specific transcripts relative to each other, but also to simultaneously measure global differences in RD (normalized ribosome density values) between samples. Graphic abstract: Global changes in translation efficiency can be detected with polysome profiling, where the proportion of polysomal ribosomes is interpreted as a proxy for ribosome density (RD) on bulk mRNA. Ribo-Seq measures changes in RD of specific mRNAs relative to bulk mRNA. The addition of a yeast-lysate, as a spike-in for normalization of read counts, allows for an absolute measurement of changes in RD.
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Project description:Expression profiling of normal NIH3T3 and transformed NIH3T3 K-ras cell lines grown for 72 hours in optimal glucose availability (25 mM glucose) or low glucose availability (1 mM). Low glucose induces apoptosis in transformed cells as compared to normal ones.
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Project description:N7-methylguanosine (m7G) modification, routinely occurring at the 5’ cap of mRNA or within tRNA and rRNA, also exists internally in mRNA. Although essential for mRNA translation as well as stress response, the “reader” protein for mRNA internal m7G modification is still unrevealed. Here, we reported that Quaking protein (QKI), especially QKI7, can selectively recognize the internal mRNA m7G decoration in the cytosol of various cell types. We identified over 1000 confident m7G-modified and QKI binding RNA targets with a conserved motif, “GANGAN (N=A/U/G)”. More strikingly, internal m7G reader QKI7 directly interacts with the SG core protein G3BP1 and can shuttle a subset of m7G-modified transcripts into SG mRNA pool under oxidative stress condition. Additionally, by sequestering mRNA within SGs, QKI7 modulates the translation efficiency of selected transcripts. Moreover, in line with the observation that doxorubicin triggers the assembly of SGs, QKI7 mediates the sensitivity of cancer cells to chemotherapy drug treatment.
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Project description:Transcriptional profiling of mouse NIH3T3 cells comparing control NIH3T3 cells transfected with a pEFm6-BRAF with cells transfected with pEFm6-BRAFV600E. Goal was to determine the effects of BRAFV600E gene transfection on global mouse NIH3T3 cells gene expression.
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