Selective mRNA sequestration by OLIGOURIDYLATE BINDING PROTEIN 1 contributes to translational control during hypoxia in Arabidopsis
ABSTRACT: 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. 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:We employ multi-step affinity purification followed by high-throughput sequencing to determine the location of EJC complexes assembled on a cellular transcriptome in Drosophila S2 cells, finding 6% of the intron-containing genes were not associated with EJCs, and within genes with multiple introns, only specific exon-exon junctions assembled an EJC. RIP-Seq, 3 samples
Project description:Hypoxia-driven alterations in the B16 melanoma cell transcriptome account for a higher metastatic potential: evidence for a role of Ero1L We analyzed transcriptomic adaptations to hypoxia/reoxygenation in B16 melanoma cells. By Ero1L over- and down-expression in vivo, we identified this ER oxidase as an actor of tumor growth and metastasis take. In vitro culture of B16 submitted to hypoxia (oxygen rate less than 1%)
Project description:Purpose: Study hypoxia induced changes in genome-wide H3K27me3 occupancy Methods: Using the MCF7 breast epithelial adenocarcinoma cell line as a model, we studied epigenomic reprogramming as a function of fluctuating oxygen tension. To this end, we combined chromatin-immunoprecipitation and deep-sequencing analysis to identify H3K27me3-marks in MCF7 cells subjected to changes in oxygenation (i.e. acute hypoxia, chronic hypoxia). Results: H3K27me3-marks showed a rapid global increase at specific sites throughout the genome under hypoxia, both genic and inter-genic. Conclusions: Our data show that oxygen availability dynamically regulates the epigenetic state of the genome. Genome-wide H3K27me3-mark profiles were generated by combining ChIP analysis with deep sequencing using Illumina GAIIx.
Project description:Plant organs are comprised of distinct cell types with unique assemblages of mRNAs. This is a collection of CEL files of mRNA profiles of the total steady-state mRNAs and polysomal mRNAs of distinct cell types of the root tip of 7-d-old Arabidopsis thaliana seedlings. The cell type specific mRNA populations are those present in ribosome-mRNA complexes. This sub-population of mRNAs was obtained by first establishing a collection of Arabidopsis lines that express a FLAG-epitope tagged ribosomal protein L18 (RPL18) directed by promoters expressed in specific cell types and regions. Four different promoter:FLAG-RPL18 lines were used. The targeted cell types and promoters included root endodermis (pSCR) and root stelar xylem and pericycle (pWOL, pSHR). A CaMV 35S promoter:FLAG-RPL18 line was used to obtain the polysomal mRNA of multiple cell types. The immunopurification of ribosome-mRNA complexes of specific cell types was accomplished by the method described in Zanetti et al. (Plant Physiology, 138, 624-635; 2005). Hybridization of the immunopurified mRNAs to the Affymetrix ATH1 DNA microarray platform and subsequent data analysis permitted the identification of transcripts that are enriched or depleted in specific cell types of root tips. The dataset includes samples from cell types from seedlings grown under control conditions and cell types of seedlings exposed to low oxygen stress (hypoxia) for 2 h. Experiment Overall Design: 20 samples, 2 conditions (2 h hypoxia stress, 2 h non-stress), 2 RNA pools (Total mRNA and polysomal mRNA), 4 promoter lines, 2 replicates
Project description:Little is known of the transcriptome of in vivo-grown pollen tubes, due to the difficulty of collection of pollen tubes elongating within the maternal gynoecium.We obtained the mRNAs undergoing translation (the translatome) of in vivo-grown pollen tubes from self-pollinated gynoecia of Arabidopsis thaliana(Col-0). Transgenic Arabidopsis plants (LAT52-HF-RPL18) harboring an epitope tagged ribosomal protein L18 driven by the pollen specific promoter (ProLAT52) were used for mRNA-ribosome complex isolation. After collection of polyribosomal (polysomal) complexes from self-pollinated (in vivo), unpollinated styles (buds), and in vitro-cultured pollen tubes, the actively translated mRNAs (the translatome) were purified, amplified to antisense RNA (aRNA). These aRNAs were hybridized to microarrays.Three independent biological replicates samples of aRNA from Bud, in vivo, and in vitro polysomal mRNA (translatomes) were hybridized to GeneChips to produce CEL files.
Project description:Since normal brain function depends upon continuous oxygen delivery and short periods of hypoxia can precondition against subsequent ischemia, this study examined the effects of brief hypoxia on the whole genome transcriptional response in adult mouse brain. Genomic expression profiling was perfromed for individual brain regions of the adult mice following the entire time course of hypoxia preconditioning. Adult C57BL/6 male mice were exposed to systemic preconditioning hypoxia (8% O2 ) for 3 hr and allowed to recover in normoxia for 24 hr. The mouse brains were removed and dissected into individual brain regions at multiple time points during the 3hr hypoxia and subsequent 24hr reoxygenation periods. Total RNA was purified from the human whole blood or individual mouse brain regions. Genomic scale gene expression was then measured with Affymetrix Mouse Expression 430 2.0 arrays.
Project description:Plant organs are comprised of distinct cell types with unique assemblages of mRNAs. This is a collection of CEL files of mRNA profiles of the total steady-state mRNAs and polysomal mRNAs of distinct cell types of the whole root and shoot of 7-d-old Arabidopsis thaliana seedlings. The cell type specific mRNA populations are those present in ribosome-mRNA complexes. This sub-population of mRNAs was obtained by first establishing a collection of Arabidopsis lines that express a FLAG-epitope tagged ribosomal protein L18 (RPL18) directed by promoters expressed in specific cell types and regions. Thirteen different promoter:FLAG-RPL18 lines were used. The targeted cell types and promoters included root atrichoblast (non-hair) epidermal cells (pGL2), root endodermis (pSCR), root stelar xylem and pericycle (pWOL, pSHR), root phloem companion cells (phloem CC) (pSUC2, pSultr2;2), root proliferating cells (pRPL11C), root cortex meristematic cells (pCO2), root cortex elongation/maturation cells (pPEP), shoot mesophyll (pRBCS), shoot epidermis (pCER5), shoot guard cells (pKAT1), shoot bundle sheath (pSultr2;2), shoot phloem CC (pSUC2) and shoot trichomes (pGL2). A CaMV 35S promoter:FLAG-RPL18 line was used to obtain the polysomal mRNA of multiple cell types. The immunopurification of ribosome-mRNA complexes of specific cell types/regions was accomplished by the method described in Zanetti et al. (Plant Physiology, 138, 624-635; 2005). Hybridization of the immunopurified mRNAs to the Affymetrix ATH1 DNA microarray platform and subsequent data analysis permitted the identification of transcripts that are enriched or depleted in specific cell types/regions of roots and shoots. The dataset includes samples from cell types/regions from seedlings grown under control conditions and cell types/regions of seedlings exposed to low oxygen stress (hypoxia) for 2 h. Experiment Overall Design: 79 samples, 2 conditions (2 h hypoxia stress, 2 h non-stress), 2 RNA pools (Total mRNA and polysomal mRNA), 2 organs, 13 promoter lines, 2-4 replicates
Project description:For identification of proteins that associate with Makorin1 (MKRN1) in RNA-dependent and RNA-independent manners, we affinity purified FLAG-tagged Makorin1 (MKRN1) from mouse embryonic stem cells constitutively expressing FLAG:MKRN1. Anti-FLAG control immunoprecipitations were performed from a FLAG vectrol control (FLAG:Ctrl) mouse embryonic stem cell line that did not express FLAG:MKRN1. Following FLAG immunoprecipitation, anti-FLAG beads from FLAG:MKRN1 and FLAG:Ctrl immunoprecipitations were split into separate tubes such that half of the beads were digested with 200Ã∞â≈ Ã≠Âµg/mL RNase A while the other half of the beads were undigested. RNase A-digested and undigested immunoprecipitates were subjected to LC-MS/MS analysis. Of the 48 RNA-related proteins previously identified to associate with FLAG:MKRN1, L1TD1, PABPC1, PABPC4, YBX1, IGF2BP1 and UPF1 were found to remain associated with FLAG:MKRN1 in the presence of RNase A.
Project description:Given that RHA regulates translation by binding a PCE located at the 5’ UTR of the target transcripts a genome-wide screen was performed to identify mRNAs that bind RHA in vivo. The results from four experiments with samples obtained in four independent RNA immunoprecipitations identified 375 transcripts that co-immunoprecipitate with FLAG RHA. Since N-terminal FLAG tagged RHA specifically co-immunoprecipitates PCE-containing mRNAs HEK293 cells were transfected with a CMV-FLAG-RHA construct. Cytoplasmic lysates were immunoprecipitated with anti-FLAG beads. RNA was extracted from the immunoprecipitate and used to probe a human Agilent expression arrays. An immunoprecipitation with cells transfected with empty FLAG plasmid was used as negative control.