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Accession IconSRP096672

Regulation of mRNA translation and subcellular location controls protein synthesis of key modulators of the DNA damage response during B cell activation [PolyRiboSeq]

Organism Icon Mus musculus
Sample Icon 157 Downloadable Samples
Technology Badge IconIllumina HiSeq 1000

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Description
Post-transcriptional regulation of cellular mRNA is essential for protein synthesis. Here we describe the importance of mRNA translational repression and mRNA subcellular location for protein expression during B lymphocyte activation and the DNA damage response. Cytoplasmic RNA granules are formed upon cell activation with mitogens, including stress granules that contain the RNA binding protein Tia1. Tia1 binds to a subset of transcripts involved in cell stress, including p53 mRNA, and controls translational silencing and RNA granule localization. DNA damage promotes mRNA relocation and translation in part due to dissociation of Tia1 from its mRNA targets. Upon DNA damage, p53 mRNA is released from stress granules and associates with polyribosomes to increase protein synthesis. Global analysis of cellular mRNA abundance and translation indicates that this is an extended ATM-dependent mechanism to increase protein expression of key modulators of the DNA damage response. Overall design: Splenic B cells from C57BL/6Babr mice were isolated and activated with LPS for 48 hours prior induction or not of DNA damage with etoposide. After 4 hours, cells were treated with cycloheximide (100 microgrames per ml) for 3 minutes. Then, cytoplasmic extracts were collected. Polysome fractionation in sucrose gradients (10-50% sucrose) was performed for isolation of mRNA associated to monosomes (fractions 4 to 7), light polysomes (fractions 8 to 10) or heavy polysomes (fractions 11 to 16). The ATM kinase inhibitor KU55933 was added 1 hour prior induction of DNA damage with etoposide.
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157
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