Rpl24b/WT polysome loading and transcript levels (Arabidopsis thaliana)
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE51474 | GEO | 2014/01/01
SECONDARY ACCESSION(S): PRJNA223281
REPOSITORIES: GEO
Similar Datasets
Project description:To understand the contribution of the RPL24B protein, a component of the large 60S ribosomal subunit, to the translation of specific mRNAs, we compared the ribosome occupancy of mRNAs in wild type Arabidopsis and the rpl24b/stv1-1 T-DNA insertion mutant. RNA was fractionated using sucrose gradients into polysomal and nonpolysomal RNAs. We also determined overall total transcript levels. We used Affymetrix ATH1 microarrays. Each plant sample was analyzed for the mRNA abundance in total mRNA, polysomal mRNA, and nonpolysomal mRNA. Three biological replicates were collected. The rpl24b mutant was compared with wild type.
2014-01-01 | E-GEOD-51474 | biostudies-arrayexpress
Project description:To understand the contribution of the poly(A)binding protein to the translation of specific mRNAs, we compared the ribosome occupancy of mRNAs in wild type Arabidopsis and pab2 pab8 double mutant seedlings. The mutants continue to express the PAB4 paralog of PABP. RNA was fractionated using sucrose gradients into polysomal and nonpolysomal RNAs. We also determined overall total transcript levels. We used Affymetrix ATH1 microarrays.
2014-01-01 | GSE51480 | GEO
Project description:To understand the role of GCN2 in regulating translation, we compared the polysome loading state and overall transcript level between Arabidopsis thaliana wild type (ecotype Landsberg erecta) and gcn2 (Genetrap line GT8359, Cold Spring Harbor Laboratory) seedlings with or without herbicide chlorosufuron treatment RNA was fractionated using sucrose gradients into polysomal and nonpolysomal RNAs. We also determined overall total transcript levels. We used Affymetrix ATH1 microarrays.
2015-12-01 | GSE52116 | GEO
Project description:To understand the contribution of the poly(A)binding protein to the translation of specific mRNAs, we compared the ribosome occupancy of mRNAs in wild type Arabidopsis and pab2 pab8 double mutant seedlings. The mutants continue to express the PAB4 paralog of PABP. RNA was fractionated using sucrose gradients into polysomal and nonpolysomal RNAs. We also determined overall total transcript levels. We used Affymetrix ATH1 microarrays. Each plant sample was analyzed for the mRNA abundance in total mRNA (T), polysomal mRNA (PL), and nonpolysomal mRNA (NP). Four biological replicates were collected for polysomes and three for total RNA. The pab2 pab8 double mutant was compared with wild type.
2014-01-01 | E-GEOD-51480 | biostudies-arrayexpress
Project description:To understand the role of GCN2 in stress response, the total transcript and translation state were compared between Arabidopsis thaliana wild type (ecotype Landsberg erecta) and gcn2 (Genetrap line GT8359, Cold Spring Harbor Laboratory) seedlings with or without herbicide chlorosufuron treatment RNA was fractionated using sucrose gradients into polysomal and nonpolysomal RNAs. We also determined overall total transcript levels. We used Affymetrix ATH1 microarrays.
2015-12-01 | GSE52117 | GEO
Project description:To understand the contribution of the k subunit of eukaryotic transcription factor 3 (eif3k) to the translation of specific mRNAs, we compared the polysome loading states and overall transcript levels of wild type Arabidopsis and the eif3k T-DNA insertion mutant by Affymetrix arrays. We analyzed the polysome loading states of wild type Arabidopsis and the eif3k mutant using the Arabidopsis Affymetrix ATH1 array. Data from 3 biological replicates were collected.
2011-03-29 | E-GEOD-28223 | biostudies-arrayexpress
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: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:To understand the contribution of the k subunit of eukaryotic transcription factor 3 (eif3k) to the translation of specific mRNAs, we compared the polysome loading states and overall transcript levels of wild type Arabidopsis and the eif3k T-DNA insertion mutant by Affymetrix arrays.
2011-03-29 | GSE28223 | GEO
Project description:Eukaryotic genomes contain various types of small non-coding RNAs such as microRNAs (miRNAs), silencing RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). Recent studies point to the presence of a more diverse array of uncharacterized small regulatory RNAs including those that are generated from mRNAs, rRNAs and tRNAs. To explore the possible involvement of tRNA-derived fragments (tRFs) in translational regulation, we fractionated 1 and 8h Drosophila embryos on sucrose density gradients and quantitatively measured by deep-dequencing the expression levels of the tRFs in the fractionated and unfractionated Drosophila embryonic cytosolic extracts. Analysis of 9,007,661 reads has revealed that tRFs, which are produced mainly from the 5’ends of a subset of tRNAs, are mostly associated with the non-polysomal fractions in Drosophila embryos. Quantitative analysis indicates that the expression levels of a subset of tRFs change temporally following the maternal-tozygotic transition in embryos. We detected non-polysomal association of tRFs in S2 cells as well. When transfected into S2 cells, the biotinylated tRFgly:GCC:5 co-fractionated with the non-polysomal complexes similar to the endogenous tRFgly:GCC:5 in embryos and S2 cells. These results suggest that the tRFs, which are much smaller in size than the stres-induced tRNA fragments, are generated selectively from a fraction of tRNAs and that they associate primarily with the non-polysomal complexes in Drosophila embryos and S2 cells.
2018-02-12 | GSE35443 | GEO