Fig 2: SDOS localizes on the ER and associates with actively translating polyribosomes. (A) Subcellular fractionation of HeLa cells into mitochondrial (Mito), cytosolic (Cyto) and microsomal (ER) fractions following 24 h induction of SDOS-eGFP. Total lysate and fractions were analyzed by WB with indicated antibodies. BiP is used as a ER marker, GAPDH as a cytosolic marker and VDAC1 as a mitochondrial marker. (B) WB analysis of TRAP1-IP from the ER fraction with SDOS, TRAP1 antibodies. Vinculin is used as internal control. (C) Ribosomal purification from HCT116 cells and HCT116 cells transfected with SDOS-myc, followed by immunoblot with ß-ACTIN (as a cytosolic marker), ßF1ATPase (as a mitochondrial marker), RPL3 (as a ribosomal marker) and SDOS antibodies. (D) Polysome profiling absorbance, measured at 254 nm, indicates the sedimentation of the particles: fractions 1 and 2 free cytosolic proteins or light complexes; fractions from 3 to 7 ribosomal subunits (60S, 40S) and monomer (80S); fractions from 8 to 12 polysomes. Proteins from each fraction were analyzed by WB with the indicated antibodies. rpL3 was used as a ribosome marker, eIF4B and eIF4E were used as markers of translation initiation complex. (E) Representative image of PLA showing the interaction of SDOS-eGFP with phosphorylated (active) ribosomal protein S6 (phospho-rpS6) in HeLa cells following 24 h induction of SDOS-eGFP by using GFP and phospho-rpS6 antibodies. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue). Cycloheximide treatment (200 µg/ml, 1 h) was used to confirm the causal role of active protein synthesis on the binding.

Fig 3: Effects of Iturin A on STb-R-restricted mTORC1 pathway in semimembranosus muscle. (A) Western blotting analysis of IGF-1R, IRS-1, p-PI3K, p-Akt, p-mTOR, p-S6K1, p-S6, p-4EBP1, and eIF4E in semimembranosus muscle shows the change of mTORC1 signaling pathway after being fed with Iturin A and STb-R. (B) Quantification of the western blotting results and the values of the relative protein level are represented as the ratio of IGF-1R/ß-actin, IRS-1/ß-actin, p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, p-S6K1/S6K1, p-S6/S6, p-4EBP1/4EBP1, and eIF4E/ß-actin. Data are presented as mean ± SEM, and the different lowercase letters above standard error bars indicate significant differences, n = 3.

Fig 4: STAU1 promotes an IMPß-mediated replacement of the CBC by eIF4E in vivo and in vitro. (A) IPs of IMPß-FLAG in HEK293T cells depleted of STAU1. n = 3. (B–D) The in vitro replacement assay. (B) A schematic diagram of the experimental procedure. For the immunopurification of FLAG-tagged proteins, the extracts of the cells transiently expressing either FLAG-GFP or IMPß-FLAG were subjected to IPs with anti-FLAG antibody–conjugated agarose beads. The bead-bound FLAG-tagged proteins were eluted by means of 3 × FLAG peptides. For immunopurification of CBC-associated reporter mRNPs, the IPs of endogenous CBP80 were carried out in the extracts of either undepleted or STAU1-depleted cells transiently expressing SL-5' intron RLuc reporter mRNAs. (C) An in vitro replacement assay using reporter mRNPs immunopurified from the undepleted cells. The bead-bound mRNPs were mixed with either immunopurified FLAG-GFP or IMPß-FLAG. After the reaction, the bead-bound mRNAs were analyzed by qRT-PCRs. n = 5; *, P < 0.05. (D) An in vitro replacement assay involving reporter mRNPs immunopurified from the STAU1-depleted cells. n = 5; #, not significant.

Fig 5: Downregulation of UPF1 or STAU1, respectively, promotes or inhibits the replacement. (A–C) The effect of UPF1 or STAU1 downregulation on the replacement. IPs of either CBP80 or eIF4E were carried out in the cytoplasmic extracts of HEK293T cells that transiently expressed SL-5' intron RLuc reporter mRNAs and were either undepleted or depleted of UPF1 or STAU1; n = 3; *, P < 0.05. (A) Relative levels of co-IPed RLuc reporter mRNAs in the IPs of CBP80. (B) Relative levels of co-IPed RLuc reporter mRNAs in the IPs of eIF4E. (C) The replacement index of the RLuc reporter mRNAs. (D) Cumulative distribution functions for the change in the relative ratio of the replacement index in STAU1-depleted cells relative to undepleted cells between non-targets and STAU1 targets. The P-value was calculated by the two-tailed Mann–Whitney U-test (n = 2).