Fig 2: Potential ubiquitination sites of KHSRP. (A) A schematic diagram of predicted ubiquitination sites in KHSRP is shown. (B, C) Indicated constructs expressing different KHSRP mutants were co-transfected with HA-Ub to 293T cells, then immunoprecipitated with Anti-FLAG M2 affinity gel and analyzed by western blotting. (D) GST-CUL3, 3×Myc-RBX1 and Myc-KLHL12 (or Myc-KLHL12ΔKelch) were co-transfected to 293T cells, and then pulled down by glutathione Sepharose beads. The E3 ligase complex was incubated with E1, E2, Biotin-Ub, and KHSRP WT or K109, 121, 122R with FLAG tags. The ubiquitination reactions were further subjected to FLAG-immunoprecipitation and were resolved by SDS-PAGE. KHSRP ubiquitination was detected by anti-streptavidin HRP antibody.

Fig 3: KLHL12 does not affect KHSRP stability. (A) KHSRP WT or KHSRP (K109, 121, 122R) with FLAG tags were transfected to RD cells, and then treated with 100 μg/ml cycloheximide (CHX) for the indicated times. Cyclin B1 was used as a positive control. Cells were lysed and analyzed by western blotting, using antibodies against FLAG, cyclin B1 and actin. (B) RD cells were co-transfected with 1 μg of FLAG-KHSRP and increasing amounts of HA-KLHL12 (0–10 μg). After 48 hours post-transfection, cell lysates were collected, resolved by SDS-PAGE and analyzed by western blot using anti-FLAG and anti-KLHL12 antibodies. (C, D) 293T cells were transfected with the indicated constructs, and then treated with 20 μM MG132 or DMSO for 4 h before cell lysis. β-catenin was used as a positive control. Cell lysates were analyzed by western blotting, using anti-FLAG, anti-Myc and anti-β-catenin antibodies.

Fig 4: KHSRP can be ubiquitinated by the KLHL12-based CUL3 complex. (A) KHSRP was found to form a complex with KLHL12 and CUL3. FLAG-KHSRP, Myc-KLHL12 and Myc-CUL3 expression vectors were co-transfected to 293T cells. The cell lysates were immunoprecipitated with Anti-FLAG M2 affinity gel, and then analyzed by western blot using anti-Myc and anti-FLAG antibodies. (B) 293T cells were co-transfected with FLAG-KHSRP, HA-Ub, Myc-KLHL12, Myc-CUL3 or Myc-CUL3ΔRBX1, and then treated with 20 μM MG132 for 4 h before harvesting. Cell lysates were immunoprecipitated with Anti-FLAG M2 affinity gel and resolved by SDS-PAGE, and KHSRP ubiquitination was detected by anti-HA antibody. The asterisk (*) represents the major modification of KHSRP. KHSRP, KLHL12, CUL3 and CUL3ΔRBX1 were visualized by individual antibodies against FLAG, KLHL12 and Myc. (C) Various constructs expressing KHSRP, Ub, KLHL12 or KLHL12 truncated form (KLHL12ΔKelch) were co-transfected to 293T cells. After 48 h, cells were treated with 20 μM MG132 for 4 h before harvesting, and lysates were then subjected to immunoprecipitation using Anti-FLAG M2 affinity gel. KHSRP ubiqitination was assessed by western blot, and KHSRP, KLHL12 and KLHL12ΔKelch were visualized using anti-FLAG and anti-Myc antibodies. (D) 293T cells were co-transfected with various indicated constructs and subsequently treated with 20 μM MG132 for 4 h before harvesting. Western blots were utilized to analyze ubiquitinated proteins. The asterisk (*) represents the major modification of KHSRP. The circle represents the major modification of KHSRP1-503. For both (C) and (D), levels of KHSRP or KHSRP1–503 with major modification were respectively normalized against levels of FLAG-KHSRP or FLAG-KHSRP1–503, and fold-changes over control reactions without KLHL12 were calculated and plotted.

Fig 5: Reduction of KHSRP ubiquitination diminishes the competitive capability of KHSRP against FUBP1. (A) RD cells overexpressing KHSRP WT or KHSRP (K109, 121, 122R) were incubated with biotinylated EV71 5′ UTR RNA. Nonbiotinylated EV71 5′ UTR RNA (lanes 2, 4–6) or yeast tRNA (lanes 8, 10–12) were used in the competition assay. Increasing amounts of (B) PTBP1, (C) FUBP1, and (D) PCBP2 recombinant proteins were added to compete with KHSRP WT or KHSRP (K109, 121, 122R) in interacting with biotinylated EV71 5′ UTR. Biotinylated EV71 5′ UTR and associated proteins were pulled down by streptavidin beads, and then analyzed by western blotting, using antibodies against FLAG, PTBP1, His and PCBP2.