Analysis of Ribosome Protected Fragments of mRNAs from yeast strains either expressing Rio1p or depleted of Rio1p
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE98333 | GEO | 2017/08/16
SECONDARY ACCESSION(S): PRJNA384766
REPOSITORIES: GEO
Similar Datasets
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115160 | GEO
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115159 | GEO
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115158 | GEO
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115161 | GEO
Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 M-NM-<g of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 M-NM-<g of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as M-bM-^IM-%5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).
2014-06-12 | E-GEOD-52799 | biostudies-arrayexpress
Project description:Purpose: Ribosome profiling and RNA-Seq were used to map the location and abundance of translating ribosomes on mouse heart and skeletal muscle transcripts. Methods: Tissue was rapidly harvested and snap-frozen to minimize bias to the pool of translating ribosomes. RNA was prepared from a single homogenate for each tissue so that starting RNA populations for both libraries were closely matched. Homogenates were not clarified before RNase digestion to avoid loss of ribosomes associated with large molecular weight complexes, and RNA-Seq libraries were prepared after rRNA subtraction to avoid positional loss of 5’ reads. Trimmed reads from 50 cycles of Illumina single-end sequencing were mapped onto a non-redundant set of 18,499 mouse protein-coding RefSeq transcripts from the nuclear genome. Results: Mapped sequence reads to myosin, actin and the giant protein titin together account for ~20% of the total mRNA-derived ribosome protected fragments (RPFs). We observed large-scale uniformity in the distribution of RPFs on the >30,000 codon titin open reading frame, from which we inferred an in vivo ribosome elongation error rate of ≤10-5. Ribosome footprints on Ttn mRNA also uncovered a novel 5’ UTR within a phylogenetically conserved intronic element that would produce ~2.35 mDa titin isoform that corresponds to the titin 'T2' band frequently described as a proteolytic artifact. Local translation efficiency across several >10 kb muscle mRNAs was also uniform, while their global translation efficiencies varied by ~20-fold suggesting initiation rate plays a major role in the translation efficiency of large mRNAs. Evidence for RPFs on 5’ UTRs was widespread with particular enrichment for ribosomes positioned at CUG codons. Comparison of global translation efficiency in cardiac and skeletal muscle revealed novel examples of tissue-specific translational control including synthesis of the myogenic factor Mef2c, and the titin-binding stress response protein Ankrd23. Conclusions: Our study represents the first detailed analysis of translation in an adult mammalian tissue generated by ribosome profiling technology. Current limitations to using ribosomal profiling in tissues include unknown perturbations to the dynamic state of translation despite rapidly harvested and snap-frozen samples. The uniform 5’ to 3’ coverage observed on individual large mRNAs and the ability to observe footprints on the extremely small phospholamban coding sequence, suggests that initiation and elongation were halted on similar time scales. More detailed examination of the positional information within CDS region requires further understanding of the bias introduced during the library preparation steps for both RPF-and RNA-Seq, as well as local biases induced as translation is arrested. Despite these qualifications, this initial view of active translation in muscle tissue highlights the potential for ribosome profiling to monitor the dynamic translation response to exercise, injury or disease pathology in animal models at a level of resolution not easily attainable with other quantitative approaches. Heart and skeletal muscle ribosome-protected fragment and RNA-Seq profiles of 10-week old C57BL/6J male mice were generated by deep sequencing using the Illumina HiSeq 2000.
2015-09-01 | E-GEOD-41246 | biostudies-arrayexpress
Project description:Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5 - 7 days to generate a completed ribosome profiling sequencing library. Ribosome profiling in cultured mammalian cells under three different footprinting conditions
2012-07-26 | E-GEOD-37744 | biostudies-arrayexpress
Project description:Translation regulation occurs largely during initiation. Currently, translation initiation can be studied in vitro, but these systems lack features present in vivo and on endogenous mRNAs. Here we develop selective 40S footprinting for visualizing initiating 40S ribosomes on endogenous mRNAs in vivo. It pinpoints where on an mRNA initiation factors join the ribosome to act, and where they leave. We discover that in human cells most scanning ribosomes remain attached to the 5’ cap. Consequently, only one ribosome scans a 5’UTR at a time, and 5’UTR length affects translation efficiency. We discover that eIF3B, eIF4G1 and eIF4E remain on translating 80S ribosomes with a decay half-length of ~12 codons. Hence ribosomes retain these initiation factors while translating short upstream Open Reading Frames (uORFs), providing an explanation for how ribosomes can re-initiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.
2020-06-25 | GSE139391 | GEO
Project description:The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo or serve as alternative initiation factors. Ribosome profiling of strains missing these factors revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3’UTR, as evidenced by peaks in the footprint data and 3’UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream ORFs (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an alternative 80S reinitiation process in 3’UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in the recycling of 40S ribosomal subunits at stop codons and translation reinitiation.
2018-08-30 | GSE108942 | GEO
Project description:Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5 - 7 days to generate a completed ribosome profiling sequencing library.
2012-07-26 | GSE37744 | GEO