Polysome profiling of Huh7.5 cells expressing HCV NS5A
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: HARINIVAS KRISHNAN
PROVIDER: E-GEOD-46273 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:In obesity, adipose tissue expands by increasing the volume of existing adipocytes (hypertrophy), increasing the number of small adipocytes (hyperplasia) or both. Much is known about transcriptional control during adipogenesis, including early events. However, translational control plays a pivotal role in many dynamic processes and we presume that it is also important for the control of adipogenesis, especially in the first hours after hormonal induction. By use of ribosome profiling we identified 43 genes that are up-regulated and 2 genes that are down-regulated during the first six hours of adipogenesis in 3T3-L1 cells. Interestingly, we found Ghrelin to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (Cdkn1c, eIF4b, Hsf1, Irf6, Myc, Plekhn1, Polr2a, Rpl18, Rpl27a, Rpl6, Rpl7a, Rps18, Rpsa, Sema3g, Tbc1d22a, Tsc22d3), form part of ribosomes (L6, L7a, L18, L27a, Sa, S18, S15a pseudogene), act on the regulation of translation (eIF4b) or transcription (Hsf1, Irf6, Myc, Tsc22d3). Others act as chaperones (Bag3, Hspa8/Hsc70, Hsp90ab1) or in other metabolic or signals transducing processes. We conclude that a moderate reorganisation of the functionality of the ribosomal machinery is a very important step for the growth and expression control at the beginning of adipogenesis. 12 samples: 4 (0h, non-polysomal fraction; 0h, polysomal fraction; 6h, non-polysomal fraction; 6h, polysomal fraction) x3 replicates
2012-03-28 | E-GEOD-29744 | biostudies-arrayexpress
Project description:Malaria is caused by Plasmodium parasites, which are transmitted via the bites of infected Anopheline mosquitoes. Midgut invasion is a major bottleneck for Plasmodium development inside the mosquito vectors as a rapidly responding immune system recognizes ookinetes and recruits killing factors from the midgut and surrounding tissues, dramatically reducing the population of invading ookinetes before they can successfully traverse the midgut epithelium. Understanding molecular details of the parasite-vector interactions requires precise measurement of nascent protein synthesis in the mosquito during Plasmodium infection. Current expression profiling primarily monitors alterations in steady-state levels of mRNA, but does not address the equally critical issue of whether the proteins encoded by the mRNAs are actually synthesized. In this study, we used sucrose density gradient centrifugation to isolate actively translating Anopheles gambiae mRNAs based upon their association with polyribosomes (polysomes). The proportion of individual gene transcripts associated with polysomes, which is determined by RNA deep sequencing, reflects mRNA translational status. This approach led to identification of 1017 mosquito transcripts that were primarily regulated at the translational level after ingestion of Plasmodium falciparum-infected blood. Caspar, a negative regulator of the NF-kappaB transcription factor Rel2, appears to be substantially activated at the translational levels during Plasmodium infection. In addition, transcripts of Dcr1, Dcr2 and Drosha, which are involved in small RNA biosynthesis, exhibited enhanced associations with polysomes after P. falciparum challenge. This observation suggests that mosquito microRNAs may play an important role in reactions against Plasmodium invasion. We analyzed both total cellular mRNAs and mRNAs that are associated with polysomes to simultaneously monitor transcriptomes and nascent protein synthesis in the mosquito. This approach provides more accurate information regarding the rate of protein synthesis, and identifies some mosquito factors that might have gone unrecognized because expression of these proteins is regulated mainly at the translational level rather than at the transcriptional level after mosquitoes ingest a Plasmodium-infected blood meal. Midguts from female Anopheles gambiae mosquitoes were dissected at around 24 h after ingestion of P. falciparum-infected blood. Mosquitoes fed on uninfected blood were used as control. A portion of the midguts were used for isolation of total cellular RNA. Extracts of the remaining midguts were fractionated over sucrose density gradients, and fifteen fractions were collected from the top of each gradient. Non-polysomal fractions, as well as polysomal fractions were combined, respectively, to obtain two RNA pools per gradient for three independent experiments. The first, last and the sample between non-polysomal and polysomal, were all discarded to ensure pure pools from each set. A fourth experiment was conducted where the polysomal and non-polysomal samples were not pooled, to be used later for qRT-PCR. The mRNA levels of individual mosquito genes in polysome (PS) fractions, nonpolysome (NP) fractions and unfractionated steady-state (total) RNA were determined using high throughput RNA deep sequencing. Each RNA pool generated 2.1-9.8 million raw read . Signal intensities of transcripts from each PS pool were compared to those of transcripts from a matching NP pool. To quantify the translational status of individual mRNA species, we define the relative PS loading [PL=P/(P+NP)] as the extent of mRNA association with polysomes. PL values were compared between the mosquitoes fed on P. falciparum-infected or -uninfected blood.
2012-09-21 | E-GEOD-38707 | biostudies-arrayexpress
Project description:The initial translational response of the yeast Saccharomyces cerevisiae in response to acetic acid-induced apoptosis was investigated by microarray profiling of mRNAs contained in polysomal fractions obtained upon 0 (used as control), 15 and 30 minutes of acetic acid treatment. The mRNA fraction thus investigated corresponds to the mRNAs capable of overcoming the inhibition of cap-mediated translation initiation.
2013-02-24 | E-MEXP-3570 | biostudies-arrayexpress
Project description:This experiment investigates the effect of mild cold-shock (32 degrees C, 24 hours) on the translatome of human HEK293 cells. After cold-shock, cells were treated with cycloheximide to block elongation, then sucrose density gradients were used to separate post-nuclear extracts into polysome and subpolysome fractions. The pooled polysomal fraction was labelled with Cy5, the pooled subpolysomal fraction was labelled with Cy3, and both were hybridised to the same array.
2017-02-23 | E-MTAB-5437 | biostudies-arrayexpress
Project description:To determine the translational status of mRNAs in wild-type and gld-1 mutant animals, we analyzed total and polysomal RNA levels in both strains by tiling arrays A total of 16 samples was analyzed (quadruplicates of wild-type_total, wild-type_polysomal, gld-1_total, gld-1_polysomal RNA respectively)
2012-06-17 | E-GEOD-33083 | 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. We analyzed the transcript levels 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-28222 | 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. 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:This SuperSeries is composed of the following subset Series: GSE28222: eif3k/WT transcript levels GSE28223: eif3k/WT polysome loading Refer to individual Series
2011-03-29 | E-GEOD-28224 | biostudies-arrayexpress
Project description:During the peri-partum period, the lung must respond to many factors with potential to impact protein synthesis via regulation of translation initiation. Microarray analysis of polysomal versus total RNA from fetal day (FD) 19, FD22 and postnatal day 1 (P1) rat lungs was used to identify genes whose association with large polysomes changed either pre- or postnatally. Experiment Overall Design: Using three independent litters at each developmental time point, we prepared lysates from FD19, FD22 and P1 (5 h after birth) rat lungs (total 9 lysates). An aliquot of each lysate was used to prepare total RNA. Remaining lysate was subjected to sucrose density gradient centrifugation to isolate large polysomes ( n>7 ribosomes/mRNA) from which we isolated polysomal RNA. Matched total and polysomal RNAs were hybridized to Affymetrix expression arrays (total 18 chips).
2009-07-11 | E-GEOD-12153 | biostudies-arrayexpress
Project description:We treated Jurkat cells for 48 hr with a sublethal dose of FK866 (5 nM) and DMSO (Mock, control treatment). RNA samples for microarrays derived from fractionated samples by sucrose gradient (sub-polysomes, polysomes), giving us the chance to perform an analysis among polysomal/subpolysomal distribution in treated or untreated cells and the possibility to identify the multi-level gene expression regulation effects of FK866. We are interested to find differentially expressed genes, in the early phase of cell response to FK866, and genes that account for a specific post-transcriptional regulation exerted by the cell in response to the drug. Keywords: polysomal profiling, translatome profiling, polysomal RNA, subpolysomal RNA, translational profiling, polysome profiling, post-transcriptional regulation, FK866, translational efficiency. Gene expression signals derived from the polysomal and subpolysomal RNA populations were compared by microarrays analysis to those obtained from total RNAs (derived from the sum of all the fractions in the polysomal gradient). Polysomal RNA, subpolysomal RNA and total RNA were isolated from Jurkat cells treated with FK866 5 nM or DMSO (mock, control treatment) for 48 hr. Cells lysates were collected from control cells (mock) and from treated cells (FK866). All experiments were run in biological quadruplicates.
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