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YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1.

Somasekharan SP, El-Naggar A, Leprivier G, Cheng H, Hajee S, Grunewald TG, Zhang F, Ng T, Delattre O, Evdokimova V, Wang Y, Gleave M, Sorensen PH - J. Cell Biol. (2015)

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.

View Article: PubMed Central - HTML - PubMed

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.

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YB-1 regulates G3BP1 translation through the G3BP1 5′ UTR. (A) mRNA transcripts bound to YB-1 were riboimmunoprecipitated (RIPed) using anti–YB-1 antibodies or normal rabbit serum (NRS) from siControl and siYB-1 kd cell lysates. Captured mRNAs were reverse-transcribed and PCR amplified using primers specific for G3BP1 or XIAP as a control. (B) YB-1–bound mRNAs were RIPed using anti–YB-1 or control anti-GRB2 antibodies from polysomes prepared from vehicle alone and arsenite-treated U2OS cells, and subjected to semiquantitative RT-PCR using G3BP1- and XIAP-specific primers. (C) Constructs containing 5′ UTR sequences of G3BP1 (black) or β-Globin (gray) fused in frame to Luciferase were used for in vitro coupled transcription translation. Increasing concentrations of recombinant YB-1 were added to the assay mixture, and luciferase activity was measured. Error bars indicate SD. (D) RNA EMSA analysis to measure direct binding of YB-1 to the full-length G3BP1 5′ UTR. Biotin-labeled full-length G3BP1 5′ UTR mixed with recombinant GST-YB-1 was subjected to EMSA. The arrowhead indicates a probe mobility shift in the presence of 0.4 µg of GST-YB-1, and enhanced intensity at 0.8 µg of GST-YB-1. A 200-fold molar excess concentration of unlabeled full-length G3BP1 5′ UTR was added to demonstrate specificity of 5′ UTR G3BP1/YB-1 complex formation. As a control, recombinant GST was used in place of GST-YB-1. The broken line indicates that intervening lanes have been spliced out. (E) The full-length 5′ UTR G3BP1 (FL, 1–171) or deletion mutants (M1, Δ105–112; M2, Δ141–171; M3, Δ99–171; and M4, Δ141–171) were cloned in frame with Luciferase and used for in vitro coupled transcription/translation assays ±0.5 pmol YB-1 as described in C. Error bars indicate SD. (F) RNA EMSA showing that YB-1 binds to the full-length (FL, 1–171) G3BP1 5′ UTR but not M3 and M4 mutants. (G) Biotin end-tagged full length or the indicated deletion mutants of the G3BP1 5′ UTR were subjected to RNA affinity chromatography from U2OS lysates using Streptavidin beads. Affinity-purified proteins were immunoblotted using anti–YB-1 antibodies. Biotin end-tagged 5′ UTR of β-Globin was used as a control. (H) Full-length G3BP1 5′ UTR or the M4 deletion mutant (Δ48–171) were transfected into siControl or siYB-1 kd U2OS cells. Lysates were prepared and subjected to RNA affinity chromatography and immunoblotted as described in G to detect 5′ UTR–bound YB-1. Untransfected cells served as controls.
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fig3: YB-1 regulates G3BP1 translation through the G3BP1 5′ UTR. (A) mRNA transcripts bound to YB-1 were riboimmunoprecipitated (RIPed) using anti–YB-1 antibodies or normal rabbit serum (NRS) from siControl and siYB-1 kd cell lysates. Captured mRNAs were reverse-transcribed and PCR amplified using primers specific for G3BP1 or XIAP as a control. (B) YB-1–bound mRNAs were RIPed using anti–YB-1 or control anti-GRB2 antibodies from polysomes prepared from vehicle alone and arsenite-treated U2OS cells, and subjected to semiquantitative RT-PCR using G3BP1- and XIAP-specific primers. (C) Constructs containing 5′ UTR sequences of G3BP1 (black) or β-Globin (gray) fused in frame to Luciferase were used for in vitro coupled transcription translation. Increasing concentrations of recombinant YB-1 were added to the assay mixture, and luciferase activity was measured. Error bars indicate SD. (D) RNA EMSA analysis to measure direct binding of YB-1 to the full-length G3BP1 5′ UTR. Biotin-labeled full-length G3BP1 5′ UTR mixed with recombinant GST-YB-1 was subjected to EMSA. The arrowhead indicates a probe mobility shift in the presence of 0.4 µg of GST-YB-1, and enhanced intensity at 0.8 µg of GST-YB-1. A 200-fold molar excess concentration of unlabeled full-length G3BP1 5′ UTR was added to demonstrate specificity of 5′ UTR G3BP1/YB-1 complex formation. As a control, recombinant GST was used in place of GST-YB-1. The broken line indicates that intervening lanes have been spliced out. (E) The full-length 5′ UTR G3BP1 (FL, 1–171) or deletion mutants (M1, Δ105–112; M2, Δ141–171; M3, Δ99–171; and M4, Δ141–171) were cloned in frame with Luciferase and used for in vitro coupled transcription/translation assays ±0.5 pmol YB-1 as described in C. Error bars indicate SD. (F) RNA EMSA showing that YB-1 binds to the full-length (FL, 1–171) G3BP1 5′ UTR but not M3 and M4 mutants. (G) Biotin end-tagged full length or the indicated deletion mutants of the G3BP1 5′ UTR were subjected to RNA affinity chromatography from U2OS lysates using Streptavidin beads. Affinity-purified proteins were immunoblotted using anti–YB-1 antibodies. Biotin end-tagged 5′ UTR of β-Globin was used as a control. (H) Full-length G3BP1 5′ UTR or the M4 deletion mutant (Δ48–171) were transfected into siControl or siYB-1 kd U2OS cells. Lysates were prepared and subjected to RNA affinity chromatography and immunoblotted as described in G to detect 5′ UTR–bound YB-1. Untransfected cells served as controls.

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.


YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1.

Somasekharan SP, El-Naggar A, Leprivier G, Cheng H, Hajee S, Grunewald TG, Zhang F, Ng T, Delattre O, Evdokimova V, Wang Y, Gleave M, Sorensen PH - J. Cell Biol. (2015)

YB-1 regulates G3BP1 translation through the G3BP1 5′ UTR. (A) mRNA transcripts bound to YB-1 were riboimmunoprecipitated (RIPed) using anti–YB-1 antibodies or normal rabbit serum (NRS) from siControl and siYB-1 kd cell lysates. Captured mRNAs were reverse-transcribed and PCR amplified using primers specific for G3BP1 or XIAP as a control. (B) YB-1–bound mRNAs were RIPed using anti–YB-1 or control anti-GRB2 antibodies from polysomes prepared from vehicle alone and arsenite-treated U2OS cells, and subjected to semiquantitative RT-PCR using G3BP1- and XIAP-specific primers. (C) Constructs containing 5′ UTR sequences of G3BP1 (black) or β-Globin (gray) fused in frame to Luciferase were used for in vitro coupled transcription translation. Increasing concentrations of recombinant YB-1 were added to the assay mixture, and luciferase activity was measured. Error bars indicate SD. (D) RNA EMSA analysis to measure direct binding of YB-1 to the full-length G3BP1 5′ UTR. Biotin-labeled full-length G3BP1 5′ UTR mixed with recombinant GST-YB-1 was subjected to EMSA. The arrowhead indicates a probe mobility shift in the presence of 0.4 µg of GST-YB-1, and enhanced intensity at 0.8 µg of GST-YB-1. A 200-fold molar excess concentration of unlabeled full-length G3BP1 5′ UTR was added to demonstrate specificity of 5′ UTR G3BP1/YB-1 complex formation. As a control, recombinant GST was used in place of GST-YB-1. The broken line indicates that intervening lanes have been spliced out. (E) The full-length 5′ UTR G3BP1 (FL, 1–171) or deletion mutants (M1, Δ105–112; M2, Δ141–171; M3, Δ99–171; and M4, Δ141–171) were cloned in frame with Luciferase and used for in vitro coupled transcription/translation assays ±0.5 pmol YB-1 as described in C. Error bars indicate SD. (F) RNA EMSA showing that YB-1 binds to the full-length (FL, 1–171) G3BP1 5′ UTR but not M3 and M4 mutants. (G) Biotin end-tagged full length or the indicated deletion mutants of the G3BP1 5′ UTR were subjected to RNA affinity chromatography from U2OS lysates using Streptavidin beads. Affinity-purified proteins were immunoblotted using anti–YB-1 antibodies. Biotin end-tagged 5′ UTR of β-Globin was used as a control. (H) Full-length G3BP1 5′ UTR or the M4 deletion mutant (Δ48–171) were transfected into siControl or siYB-1 kd U2OS cells. Lysates were prepared and subjected to RNA affinity chromatography and immunoblotted as described in G to detect 5′ UTR–bound YB-1. Untransfected cells served as controls.
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fig3: YB-1 regulates G3BP1 translation through the G3BP1 5′ UTR. (A) mRNA transcripts bound to YB-1 were riboimmunoprecipitated (RIPed) using anti–YB-1 antibodies or normal rabbit serum (NRS) from siControl and siYB-1 kd cell lysates. Captured mRNAs were reverse-transcribed and PCR amplified using primers specific for G3BP1 or XIAP as a control. (B) YB-1–bound mRNAs were RIPed using anti–YB-1 or control anti-GRB2 antibodies from polysomes prepared from vehicle alone and arsenite-treated U2OS cells, and subjected to semiquantitative RT-PCR using G3BP1- and XIAP-specific primers. (C) Constructs containing 5′ UTR sequences of G3BP1 (black) or β-Globin (gray) fused in frame to Luciferase were used for in vitro coupled transcription translation. Increasing concentrations of recombinant YB-1 were added to the assay mixture, and luciferase activity was measured. Error bars indicate SD. (D) RNA EMSA analysis to measure direct binding of YB-1 to the full-length G3BP1 5′ UTR. Biotin-labeled full-length G3BP1 5′ UTR mixed with recombinant GST-YB-1 was subjected to EMSA. The arrowhead indicates a probe mobility shift in the presence of 0.4 µg of GST-YB-1, and enhanced intensity at 0.8 µg of GST-YB-1. A 200-fold molar excess concentration of unlabeled full-length G3BP1 5′ UTR was added to demonstrate specificity of 5′ UTR G3BP1/YB-1 complex formation. As a control, recombinant GST was used in place of GST-YB-1. The broken line indicates that intervening lanes have been spliced out. (E) The full-length 5′ UTR G3BP1 (FL, 1–171) or deletion mutants (M1, Δ105–112; M2, Δ141–171; M3, Δ99–171; and M4, Δ141–171) were cloned in frame with Luciferase and used for in vitro coupled transcription/translation assays ±0.5 pmol YB-1 as described in C. Error bars indicate SD. (F) RNA EMSA showing that YB-1 binds to the full-length (FL, 1–171) G3BP1 5′ UTR but not M3 and M4 mutants. (G) Biotin end-tagged full length or the indicated deletion mutants of the G3BP1 5′ UTR were subjected to RNA affinity chromatography from U2OS lysates using Streptavidin beads. Affinity-purified proteins were immunoblotted using anti–YB-1 antibodies. Biotin end-tagged 5′ UTR of β-Globin was used as a control. (H) Full-length G3BP1 5′ UTR or the M4 deletion mutant (Δ48–171) were transfected into siControl or siYB-1 kd U2OS cells. Lysates were prepared and subjected to RNA affinity chromatography and immunoblotted as described in G to detect 5′ UTR–bound YB-1. Untransfected cells served as controls.
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.

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.

View Article: PubMed Central - HTML - PubMed

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
Related in: MedlinePlus