Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Polysome purified mRNA in control and RACK1 silenced in human hepatocarcinoma cells

ABSTRACT: Fighting viral infections is hampered by the scarcity of viral targets and their variability resulting in development of resistance. Viruses depend on cellular molecules for their life cycle, which are attractive alternative targets, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection indicating that its function is conserved among distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a new target for the development of broad antiviral intervention. 4 Controls 4 RACK1 silenced cells

ORGANISM(S): Homo sapiens

SUBMITTER: Catherine Schuster

PROVIDER: E-GEOD-60374 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

RACK1 controls IRES-mediated translation of viruses.

Majzoub Karim K Hafirassou Mohamed Lamine ML Meignin Carine C Goto Akira A Marzi Stefano S Fedorova Antonina A Verdier Yann Y Vinh Joëlle J Hoffmann Jules A JA Martin Franck F Baumert Thomas F TF Schuster Catherine C Imler Jean-Luc JL

Cell 20141101 5

Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further ...[more]

PMID: 25416947

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Project description:Integrated breeding strategies are used to increase both the yield potential and stability of crops. Most of these approaches have a direct genetic basis. The utility of epigenetics in breeding to improve complex traits such as yield and stress tolerance is not clear. A better understanding of the status of the epigenome and its contribution to the agronomic performance would help in developing strategies to incorporate the epigenetic component of complex traits in breeding,Starting from isogenic canola lines, epilines were generated by selecting recursively during three generations for lines with a higher energy use efficiency and drought tolerance. These epilines were more energy use efficient, drought tolerant, high nitrogen use efficient, and higher yielding under suboptimal conditions. Moreover, these characteristics were transgenerational inheritable. Transcriptome comparison with a line selected for energy use efficiency only revealed common differentially expressed genes related to the onset of signaling events regulating stress tolerance. Genes related to salt, osmotic, abscisic acid and drought were specifically differentially expressed in the drought tolerant epilines. The status of the epigenome, scored as differential trimethylation of lysine 4 of histone 3, supports the energy use efficient and drought tolerant phenotype by facilitating transcription of the genes that are found to be differentially expressed.From these results it can be concluded that the epigenome can be shaped by selection to increase yield and stress tolerance. This acquired knowledge will support further development of strategies to incorporate epigenetics in breeding. SRA study accession: SRP052946, Bioproject: PRJNA273932. Two canola (Brassica napus L. spp. oleifera (Metzg) Sinsk. f. biennis) epilines with low respiration and high NAD(P)H content were selected. Grinded leaf material from 26 day-old control and both epilines was collected in triplicate and served as input for deep sequencing on Illumina GAIIx.

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).

Dataset Information

Polysome purified mRNA in control and RACK1 silenced in human hepatocarcinoma cells

Publications

RACK1 controls IRES-mediated translation of viruses.

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