Figure 3.
Figure 3. 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.

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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