Extensive translation of small Open Reading Frames revealed by Poly-Ribo-Seq
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
SUBMITTER: Juan Pablo Couso
PROVIDER: E-GEOD-60384 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
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.
2014-09-11 | GSE60384 | 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:We present a genome-wide assessment of the translation of small open reading frames (smORF) in Drosophila melanogaster mRNAs, using ribosomal profiling of polysomal fractions in three contiguous temporal windows, which encopass all of embryogenesis. We also performed the same protocol using S2 cells. In this way, mRNAs bound by multiple ribosomes can be isolated and distinguished from mRNAs bound by sporadic, putatively non-productive single ribosomes or ribosomal subunits.
2020-03-27 | GSE147619 | GEO
Project description:The sequence of gene regulatory events that drive neonatal germ cell development in the mammalian testis is not yet clear. We assessed changes in mRNA utilization in the neonatal testis at 1 and 4 dpp, times when the testis contains quiescent gonocytes (1 dpp) and proliferating spermatogonia (4 dpp). There are not thought to be major changes in the nature or number of somatic cells over that interval. We used microarrays to detail the global expression levels of mRNA distribution between non-translating mRNAs and efficiently translating mRNAs during testis development at 1 dpp and 4 dpp Extracts of mouse testes from 1 dpp and 4 dpp CD-1 mice were fractionated over a sucrose gradient in triplicate. Gradients were fractionated using an ISCO gradient fractionation system equipped with a UA-6 detector. The position of the 80S peak defined the boundary between translating and non-translating mRNAs. Pooled non-polysomal (non-translating; fractions 1-3) and heavy polysomal (efficiently translating, fractions 7-13) RNAs were isolated from sucrose gradient fractions for hybridization on Affymetrix 420 2.0 microarrays.
2013-07-27 | E-GEOD-49283 | biostudies-arrayexpress
Project description:Liver samples were lysed (15 mM Tris-HCl (pH 8.0), 300 mM NaCl and 15 mM MgCl2, plus inhibitors (1 mg/ml heparin 100 µg/ml cycloheximide and 80 U RNAsin)), added to a 10-50% sucrose gradient and ultracentrifuged at 182,000x g for 2 hours. The gradient was aliquotted into 16 1ml fractions and added to Tri reagent (Sigma). RNA was extracted as per the manufacturer's instructions and then sub-pooled into fractions corresponding to monosomes, light polysomes, medium polysomes and heavy polysomes, according to ribosomal density (more ribosomal occupancy = higher translational activity = heavier, and therefore, located in the more dense fractions). Microarray analysis was performed by hybridising control (vehicle treated) monosomes against test (high-dose treated) monosomes on one microarray, control light polysomes against test light polysomes on a second microarray, and so forth for each sub-pool of fractions. Following microarray analysis there were a maximum of four values for each mRNA, corresponding to the proportional representation of that mRNA within each sub-pool of fractions. By calculating the change in values, i.e. degree of slope, across the monosomal region, the light polysomal region, the medium polysomal region and the dense polysomal region it was possible to determine any translational change in activity. In addition, the overall transcriptional change could be analysed for each mRNA. Dual colour microarrays performed on n = 3 samples, implementing a dye swap design. Control samples were treated with vehicle only. The treated samples had 1120mg/kg of the compound (Tetraethyl[(3-hydroxy-2-pyridyl)amino]-methanediphosphonate) for 15 days.
2012-06-20 | E-GEOD-38807 | biostudies-arrayexpress
Project description:Cotranslational targeting into the endoplasmic reticulum (ER) by the Signal Recognition Particle (SRP) is a key event determining polypeptide fate in eukaryotic cells. Here, we globally define the principles and mechanisms of SRP binding and ER targeting in vivo. Cotranslational targeting through SRP is the dominant route into the ER for all secretory proteins, regardless of targeting signal characteristics. Cytosolic SRP functions in a pioneer translation round that builds a membrane-resident mRNAs pool, explaining how low SRP levels suffice for the secretory load. SRP does not induce an elongation arrest; consequently, kinetic competition between targeting and translation elongation dictates which substrates are translocated post-translationally. Unexpectedly, SRP binds most secretory ribosomal complexes before targeting signals are synthesized. We show non-coding mRNA elements can promote signal-independent SRP pre-recruitment. Our study defines the complex kinetic interplay between elongation and determinants in the polypeptide and mRNA modulating SRP-substrate selection and membrane targeting in vivo. Ribosome profiling (RiboSeq) and RNA-seq of subcellular fractions of ribosomes. Soluble and membrane bound ribosomes are separated by centrifugation, and SRP-bound ribosomes are immunoprecipitated from the soluble fraction. Polysomes and monosomes are separated by sucrose gradient ultracentrifugation.
2016-08-03 | E-GEOD-74393 | biostudies-arrayexpress
Project description:Translation complexes are stabilised in vivo using formaldehyde. Cells are lysed, and cell extracts are treated with RNase I to produce protected RNA fragments (FPs or footprints). Extracts are then run through sucrose gradients to separate small ribosomal subunits and full ribosomes. FPs are isolated from each of these fractions and analysed by sequencing. Note this is a modified version of ribo-seq (a.k.a. ribosome profiling)
2022-12-02 | E-MTAB-12264 | biostudies-arrayexpress
Project description:Elavl1/HuR is a ubiquitous and conserved RNA-binding protein that binds to a U-rich RNA motif that shuttles between nucleus and cytoplasm. In epithelia, the elevated expression of HuR assumingly promotes degeneration and cancer suggesting that its generic suppression may provide clinical benefits. In this study we focused on biological and clinical functions of HuR in intestinal epithelial cells and we presented evidence that changes in HuR levels induce polarized distortions in these cells to support different pathologic outcomes. For translation profiling cytoplasmic fractions of CMT93 sublines, containing monosomes and polysomes were separated as in (Katsanou et al., 2009). Fractions containing monosomes and polysomes were collected using a retriever 500 fraction collector (Teledyne Isco). Monosomal and polysomal fractions were pooled and total RNA was extracted from each fraction using Trizol reagent (Invitrogen). For micorarrays, RNAs were further purified via columns (Qiagen). Biotinylated complementary RNAs,(cRNAs) were hybridized onto Affymetrix Gene Mo-Gene-1.0 Chip in accordance to the protocols of the Genomics Unit of BSRC “Alexander Fleming”. (http://www.fleming.gr/facilities/genomics).
2020-07-01 | E-MTAB-4016 | biostudies-arrayexpress
Project description:Purpose: mRNA translation into protein is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants has yet to be systematically studied. Using high-throughput sequencing (RNA-seq), we have measured cellular levels of mRNAs and ncRNAs, and their isoforms, in lymphoblast cell lines (LCL) and in polysomal fractions, the latter shown to yield strong correlations of mRNAs with expressed protein levels. Analysis of allelic RNA ratios at heterozygous SNPs served to reveal genetic factors in ribosomal loading. Methods: RNA-seq was performed on cytosolic extracts and polysomal fractions (3 ribosomes or more) from three lymphoblastoid cell lines. As each RNA fraction was amplified (NuGen kit), and relative contributions from various RNA classes differed between cytosol and polysomes, the fraction of any given RNA species loaded onto polysomes was difficult to quantitate. Therefore, we focused on relative recovery of the various RNA classes and rank order of single RNAs compared to total RNA. Results: RNA-seq of coding and non-coding RNAs (including microRNAs) in three LCLs revealed significant differences in polysomal loading of individual RNAs and isoforms, and between RNA classes. Moreover, correlated distribution between protein-coding and non-coding RNAs suggests possible interactions between them. Allele-selective RNA recruitment revealed strong genetic influence on polysomal loading for multiple RNAs. Allelic effects can be attributed to generation of different RNA isoforms before polysomal loading or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Several variants and genes identified by this approach are also associated with RNA expression and clinical phenotypes in various databases. Conclusions: These results provide a novel approach using complete transcriptome RNA-seq to study polysomal RNA recruitment and regulatory variants affecting protein translation. cells from 3 samples were grown to 5x105 cells/mL density in T75 tissue culture flask and harvested, total RNA and polysome bound RNA was sequenced by Ion Proton
2015-06-30 | E-GEOD-66135 | biostudies-arrayexpress
Project description:Several genome-wide transcriptome analyses that focused on p53-induced cellular responses in many cellular contexts have continued to expand the already vast p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses we performed translatome analysis by polysomal profiling on MCF7 cells treated with Doxorubicin and Nutlin-3a. A comparison between the transcriptome and the translatome revealed a large number of uncoupled genes, whose transcription changes did not correlate with translation changes. Overall, we establish p53 as a master regulator of translational control and identify many p53 target genes affecting translation that can contribute to p53-dependent cellular responses. Keywords: p53, transcriptome, translatome, polysomal RNA, subpolysomal RNA, uncoupling, Doxorubicin, Nutlin-3a Total RNA (tot) was extracted from MCF7 vector cells after 16h of treatment with Doxorubicin (1.5uM) and Nutlin-3a (10uM) or DMSO (solvent, as control treatment). Polysomal profiling was performed after the same conditions. We collected all subpolysomal mRNA fractions (sub) and the polysomal ones (pol) after sucrose gradient fractionation of cytoplasmic lysates to analyze separately mRNAs that are not actively translated from those that are considered in active translation, respectively. Experiments were performed in three biological replicates.
2014-04-17 | E-GEOD-50650 | biostudies-arrayexpress