Fig. 5: TRMP regulates p27 protein levels by competing p27 mRNA for PTBP1 binding.

a Lysates from A549 cells were incubated with either sense or antisense biotin-labeled DNA oligomers corresponding to TRMP, followed by the pull-down experiments using streptavidin-coated beads. The pull-downed proteins were separated by SDS-PAGE and visualized by Coomassie brilliant blue staining. The separated proteins were analyzed by mass spectrometry. PTBP1 was identified as a potential TRMP-binding protein. The PTBP1 peptide sequences obtained by MS are shown in Supplementary Figure S4A. b Lysates from HEK293T cells expressing Flag or Flag-PTBP1 were immunoprecipitated with anti-Flag antibody. Eluted products from the immunoprecipitates were used for examining TRMP and PTBP1 levels by semi-quantitative RT-PCR and western blot analyses, respectively. The same eluted products were also subjected to real-time RT-PCR analysis to examine TRMP levels. Data shown are mean ± SD (n = 3). c Lysates from A549 cells were incubated with either sense or antisense biotin-labeled DNA oligomers corresponding to TRMP, followed by the pull-down experiments using streptavidin-coated beads. The pull-downed complexes were analyzed by western blotting with anti-PTBP1 and anti-GAPDH antibodies. The same complexes were also subjected to real-time RT-PCR analysis to confirm the enrichment of TRMP by the antisense DNA oligomer, as shown in Supplementary Figure S4B. d Lysates from A549 cells were incubated with in vitro synthesized biotin-labeled TRMP or its antisense RNA, followed by the pull-down experiments using streptavidin-coated beads. The pull-downed complexes were analyzed by western blotting with anti-PTBP1 and anti-GAPDH antibodies. e In vitro synthesized TRMP was incubated with purified recombinant Flag-PTBP1 bound with M2 beads. After incubation and extensive washing, the beads-bound RNAs were eluted as templates for RT-PCR analysis. f HEK293T cells expressing control shRNA, TRMP shRNA#1, or TRMP shRNA#2 were transfected with or without Flag-PTBP1 as indicated. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag antibody. RNAs present in the input and immunoprecipitates were analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3). The input and immunoprecipitates were also analyzed by western blotting with anti-Flag antibody. * and *** indicate p < 0.05 and p < 0.001, respectively. g HEK293T cells were transfected with plasmids expressing Flag-PTBP1 and TRMP RNA in the indicated combination. Twenty-four hours later, cell lysates were immunoprecipitated with anti-Flag antibody, followed by real-time RT-PCR analysis to examine p27 mRNA levels in the immunoprecipitates. Data shown are mean ± SD (n = 3). ** indicates p < 0.01. h Purified Flag-PTBP1 proteins bound with M2 beads were incubated with in vitro synthesized p27 5′UTR (−447 to −1) in the absence or presence of different doses of in vitro transcribed TRMP. After incubation and extensive washing, the beads-bound RNAs were eluted as templates for RT-PCR analysis. i Shown is the luciferase reporter plasmid containing the p27 5′-UTR (−447 to −1) used in this study. j HEK293T cells expressing control shRNA, TRMP shRNA#1, or TRMP shRNA#2 were transfected with the p27 5′-UTR luciferase reporter plasmid. Twenty-four hours after transfection, the reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3). ** indicates p < 0.01. k HEK293T cells over-expressing either control RNA or TRMP were transfected with the p27 5′-UTR luciferase reporter plasmid. Twenty-four hours after transfection, the reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3). ** indicates p < 0.01. l A549 cells were infected with lentiviruses encoding control shRNA, PTBP1 shRNA, TRMP shRNA#1, and TRMP shRNA#2 in the indicated combinations. Forty-eight hours after infection, cell lysates were subjected to western blot analysis with anti-p27, anti-PTBP1, and anti-GAPDH antibodies. The band intensities were quantified by using ImageJ software. The ratio of p27 to GAPDH is presented in Supplementary Figure S4D