Project description:7d-old WT ler seedlings were submitted to 12h of non-stress (air) or hypoxia-stress treatment under low light conditions (45 uM m-2 s-2), and Total and Large Polysome RNA from both treatments were extracted and hybridized against Affymetrix genome chips. Values were used to evaluate changes in transcript abundance and transcript association with large polysomal complexes. TABLE 1 - Comparison between transcript abundance in non-stress and hypoxia-stress conditions. TABLE 2 - Comparison between transcript abundance in large polysome complexes (5 or more ribosomes per mRNA) in non-stress and 12 hypoxia-stress conditions. TABLE 3 - Proportion of total transcript that is associated with large polysome complexes (polysome loading) under non-stress conditions. Raw data (not normalized); TABLE 4 - Proportion of total transcript that is associated with large polysome complexes (polysome loading) under 12h of hypoxia-stress. Raw data (not normalized)
Project description:7d-old WT ler seedlings were submitted to 12h of non-stress (air) or hypoxia-stress treatment under low light conditions (45 uM m-2 s-2), and Total and Large Polysome RNA from both treatments were extracted and hybridized against Affymetrix genome chips. Values were used to evaluate changes in transcript abundance and transcript association with large polysomal complexes. TABLE 1 - Comparison between transcript abundance in non-stress and hypoxia-stress conditions. TABLE 2 - Comparison between transcript abundance in large polysome complexes (5 or more ribosomes per mRNA) in non-stress and 12 hypoxia-stress conditions. TABLE 3 - Proportion of total transcript that is associated with large polysome complexes (polysome loading) under non-stress conditions. Raw data (not normalized) TABLE 4 - Proportion of total transcript that is associated with large polysome complexes (polysome loading) under 12h of hypoxia-stress. Raw data (not normalized) Keywords: time-course
Project description:Large-scale identification of transcripts with large cross-genotype differences in abundance provides a genome-wide approach to identify natural variation in gene function. Transcript profiling was performed in 20 Arabidopsis accessions at dusk, dawn and after a 6h extension of the night. Individual genes with a high variance in transcript abundances across accessions were identified by inspection at each individual time point and by ANOVA across all three time points. Up to 40% of genes show significant changes in transcript abundance between accessions, with over-enrichment for biotic resistance, including pathogenesis resistance proteins and glucosinolate metabolism, cell wall modification, minor carbohydrate metabolism, amino acid degradation, brassinosteroid metabolism, redox and abiotic stress and underrepresentation of photosynthesis, DNA synthesis, RNA processing, regulation of transcription, protein synthesis and protein targeting. Some functional classes were enriched at all three time points. Genes related to carbon starvation-responses were enriched specifically at dawn, when their transcript abundance varied independently of carbohydrate levels, pointing to genetic variation in the sensitivity with which low carbon is sensed. Cross-accession correlation networks were generated at each time point to identify sets of genes whose transcripts show coordinated change in abundance between accessions. A cluster that was highly enriched for cold-response genes was found at dusk and after an extension of the night. Another cluster found at all three time points was highly enriched for glucosinolate biosynthesis, and correlated with glucosinolate content. We conclude that genes involved in stress and defense related processes show especially large and coordinated natural variation in transcript abundance.
Project description:Low oxygen stress dynamically regulates the translation of cellular mRNAs as a means of energy conservation in seedlings of Arabidopsis thaliana. Most of the highly hypoxia-induced mRNAs are recruited to polysomes and actively translated, whereas other cellular mRNAs become translationally inactive and are either targeted for stabilization or degradation. Here we identify the involvement of OLIGOURIDYLATE BINDING PROTEIN 1 (UBP1), a triple RNA Recognition Motif protein, in dynamic and reversible aggregation of translationally repressed mRNAs during hypoxia. Mutation or downregulation of UBP1C interferes with seedling establishment and reduces survival of low oxygen stress. By use of messenger ribonucleoprotein immunopurification, we show that UBP1C constitutively binds a subpopulation of mRNAs characterized by U-rich 3’-untranslated regions under normoxic conditions. During hypoxia, UBP1C association with non-U-rich mRNAs is enhanced concomitant with its aggregation into microscopically visible cytoplasmic foci, referred to as UBP1 stress granules (SGs). This UBP1C-mRNA association occurs as global levels of protein synthesis decline. Upon reoxygenation, rapid UBP1 SG disaggregation coincides with the return of the stabilized mRNAs to polysomes. The mRNAs that are highly induced and translated during hypoxia largely circumvent UBP1C sequestration. Thus, UBP1 is established as a component of dynamically assembled cytoplasmic mRNPs that sequester mRNAs that are poorly translated during a transient low energy stress.
Project description:Low oxygen stress dynamically regulates the translation of cellular mRNAs as a means of energy conservation in seedlings of Arabidopsis thaliana. Most of the highly hypoxia-induced mRNAs are recruited to polysomes and actively translated, whereas other cellular mRNAs become translationally inactive and are either targeted for stabilization or degradation. Here we identify the involvement of OLIGOURIDYLATE BINDING PROTEIN 1 (UBP1), a triple RNA Recognition Motif protein, in dynamic and reversible aggregation of translationally repressed mRNAs during hypoxia. Mutation or downregulation of UBP1C interferes with seedling establishment and reduces survival of low oxygen stress. By use of messenger ribonucleoprotein immunopurification, we show that UBP1C constitutively binds a subpopulation of mRNAs characterized by U-rich 3M-bM-^@M-^Y-untranslated regions under normoxic conditions. During hypoxia, UBP1C association with non-U-rich mRNAs is enhanced concomitant with its aggregation into microscopically visible cytoplasmic foci, referred to as UBP1 stress granules (SGs). This UBP1C-mRNA association occurs as global levels of protein synthesis decline. Upon reoxygenation, rapid UBP1 SG disaggregation coincides with the return of the stabilized mRNAs to polysomes. The mRNAs that are highly induced and translated during hypoxia largely circumvent UBP1C sequestration. Thus, UBP1 is established as a component of dynamically assembled cytoplasmic mRNPs that sequester mRNAs that are poorly translated during a transient low energy stress. Immunoprecipated RNA associated with Arabidopsis UBP1C (IP) was compared with total cellular RNA from light (L), mock dark (D), 2 h hypoxia, and 2 h hypoxia + 20 min reoxygenation treated samples with duplicate hybridizations to the Affymetrix ATH1 Genechip array.
Project description:A Kinetic Analysis of Auxin-mediated Changes in Transcript Abundance in Arabidopsis Reveals New Mediators of Root Growth and Development
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.
Project description:The goal of this experiment was to investigate the early transcript changes (6h) induced by hypoxia treatment in mesophyll protoplasts. A single pair (control & hypoxia) of GeneChips® was used to confirm that hypoxia treatment altered the expression of an overlapping set of genes controlled by KIN10 (At3g01090) in Arabidopsis mesophyll protoplasts. Keywords: KIN10, KIN11, darkness, hypoxia, starvation, stress, sugar signalling, Arabidopsis, SnRK1
Project description:RNA helicase A (RHA) binds its target transcripts at the post-transcriptional control element (PCE) located in the 5â?? untranslated region (UTR). This interaction represents an â??RNA switchâ?? that regulates protein synthesis. Down regulation of RHA by siRNAs was used to identify transcripts with RHA-dependent translation. Reduced accumulation of RNA in polysomes was monitored with microarrays. Changes in cytoplasmic RNA steady state abundance was monitored as well. Sixty nine genes exhibit decreased transcript polysome association when subjected to RHA downregulation. A majority of the transcripts that experienced a reduction in the polysome fraction had no significant change in their cytoplasmic abundance (45 genes). Keywords: gene expression array-based Cytoplasmic lysates of cells treated with RHA targeted or non-silencing control siRNAs were separated by sucrose density gradient centrifugation. Ribosomal RNA profiles were generated, fractions containing polysomes were collected, and RNA was extracted.