Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identification of global alteration of translational regulation in glioma in vivo

ABSTRACT: Post-transcriptional regulation of gene expression contributes to the protein output of a cell, however, methods for measuring translational regulation in complex in vivo systems are lacking. Here, we describe a sensitive method for measuring translational regulation in defined cell populations from heterogeneous tissue in vivo. We adapted the translating ribosome affinity purification (TRAP) methodology to measure the relative occupancy of individual mRNA transcripts in translating ribosomes in the Olig2-positive tumor cell population in a genetically engineered mouse model (GEM) of glioma. Global measurement of paired ribosome-bound and total cellular mRNA populations from tumor cells in vivo identified a broad distribution of relative ribosome occupancies amongst mRNA species that was highly reproducible across biological samples. Comparison of the translation state of glioma cells to non-transformed oligodendrocyte progenitor cells in normal brain identified global alteration of translation in tumor, and specifically of genes involved in cell division and synthetic metabolism. Furthermore, investigation of alteration in steady state translational efficiencies upon loss of PTEN, one of the most frequently mutated and deleted tumor suppressors in glioma, identified differential translation of proteins involved in cellular respiration, canonically regulated by PI3K/Akt signaling, and cellular glycosylation profiles, deregulation of which is known to be associated with tumor progression. Application of the translation efficiency profiling method described here to other biological contexts and conditions would extend our knowledge of the scope and impact of this important mode of gene regulation in complex in vivo systems. Total RNA and ribosome-bound RNA from Olig2+ cells in normal mouse brain and a mouse model of glioma was collected, microarray and compared.

ORGANISM(S): Mus musculus

SUBMITTER: Karim Helmy

PROVIDER: E-GEOD-38591 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To study the physiological role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2tm3(cre)Bhr strain to target deletion of Wnt4 to granulosa cells. Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had significantly reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that, while Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty, they had only 25.2% of the normal number of healthy antral follicles. Some Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RTPCR analyses of Wnt4flox/-;Amhr2tm3(cre)Bhr/+ granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1 and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 also regulates a series of genes involved in late follicle development and the cellular stress response via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development, and may act by regulating granulosa cell functions including steroidogenesis. Experiment Overall Design: Granulosa cells were obtained from 20-26 day-old mice of various genotypes 48h after eCG treatment, using the needle puncture method as previously described. Cells were then infected with adenoviruses to express eGFP or Cre, or overexpress WNT4 in serum-free medium, and subsequently harvested for RNA extraction as described below. Preliminary experiments demonstrated that an infection efficiency of >80% could be obtained at an MOI of ~50 (as determined by analysis of fluorescent signal in Ad-eGFP-infected cells) and that the Ad-Cre and Ad-WNT4 viruses produced efficient recombination of the floxed Ctnnb1 alleles and robust WNT4 overexpression, respectively. Microarray analyses were done using triplicate RNA samples from each adenoviral treatment described above, and using mouse expression set 430 microarrays (Affymetrix, Santa Clara, CA).

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Identification of global alteration of translational regulation in glioma in vivo

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