YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1.
Bottom Line: YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro.Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models.Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.
Affiliation: Department of Pathology and Laboratory Medicine and Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada.Show MeSH
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Mentions: Because YB-1 binds RNA and is a known translational regulator, we hypothesized that its effects on G3BP1 synthesis are through translational activation of G3BP1 mRNAs. To examine this, we first assessed if YB-1 physically interacts with G3BP1 transcripts in U2OS cells. We immunoprecipitated YB-1–associated mRNAs from cell extracts using YB-1 antibodies or control normal rabbit serum (NRS), and then assayed for the presence of YB-1–bound G3BP1 or control XIAP mRNAs using semiquantitative RT-PCR. As shown in Fig. 3 A, G3BP1 RT-PCR products were strongly enriched in siControl cell lysates after immunoprecipitation with anti–YB-1 but not nonspecific antibodies, whereas this enrichment was lost in siYB-1 cell immunoprecipitates. Next, to show that YB-1 associates with actively translated G3BP1 messages, we performed sucrose gradient centrifugation to isolate polysomal fractionated (ribosome-bound) mRNAs, as described previously (Evdokimova et al., 2009), followed by immunoprecipitation with anti–YB-1 antibodies to pull down YB-1 and its bound transcripts from polysomal fractions. This demonstrated that significant amounts of YB-1 are present in polysomes under both ambient and arsenite stress conditions, using RPS6 as a known polysome-associated control protein (Fig. S4 A). Moreover, semiquantitative RT-PCR showed that YB-1 associates with G3BP1 mRNAs in polysomes from U2OS cells, under both ambient and arsenite-treated conditions (Fig. 3 B). These results indicate that YB-1 interacts with actively translated G3BP1 mRNAs in cells even under ambient conditions, and that a pool of YB-1 remains available in polysomes in association with G3BP1 transcripts under arsenite stress.
Affiliation: Department of Pathology and Laboratory Medicine and Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada.