Fig 2: G3BP1 regulates the stability of PITX1 mRNA through its interaction with rG4 structures. (A) RT-qPCR results showing the half-lives of PITX1 mRNA in 293T cells, G3BP1-/- cells, PDS-treated 293T cells, and PDS-treated G3BP1-/- cells. (B) Bar chart showing the half-lives calculated from the above RT-qPCR results. (C) rG4-forming sequences in the 3'-UTR of PITX1 mRNA and their corresponding mutants. (D) Relative firefly luciferase mRNA levels (normalized to the level of renilla luciferase mRNA) and (E) relative firefly luciferase activities (normalized to renilla luciferase activity) in 293T cells, G3BP1-/- cells and PDS-treated 293T cells expressed from PITX1-WT or PITX1-3Qm plasmid. Error bars represent S.E.M. (n = 3). The P values were calculated by using unpaired, two-tailed Student's t-test. ns, P > 0.05; **, 0.001 = P < 0.01; ***P < 0.001.

Fig 3: G3BP1 binds directly and selectively with rG4 structures. Fluorescence anisotropy for measuring the binding affinities of G3BP1 protein toward rG4 structures derived from PITX1 (A) and NRAS (B) mRNAs and their corresponding mutants (rM4s). Error bars represent S.D. (n = 3). (C) Western blot images and quantitative results obtained from in vitro pull-down of G3BP1 protein from whole-cell protein lysates with the use of biotin-labeled PITX1 rG4 and rM4 probes. Error bars represent S.E.M. (n = 3). (D) A schematic diagram depicting the domain structure of G3BP1 protein. (E–H) EMSA for monitoring the interactions between the four truncated forms of G3BP1 with PITX1 rG4 probe. (I) Fluorescence anisotropy for measuring the binding affinities of G3BP1-RRM-RGG with PITX1 rG4 and rM4. Error bars represent S.D. (n = 3). The P values were calculated by unpaired, two-tailed Student's t-test. ***P < 0.001.