Fig 1: PDAP1 is required for HAV IRES-mediated translation.(A) (Left) Quasi-enveloped (eHAV) and naked virus (nHAV) attached to control sgCtrl and PDAP1-KO1.4 cells at 4°C, measured by RT-PCR. (Right) Cell uptake of HAV at 37°C. GE, genome equivalent. (B) FLuc expressed by sub-genomic HAV replicon RNA. GAA, lethal 3Dpol mutation. (C) HAV RNA decay in sgCtrl and PDAP1-KO1.4 cells electroporated with 18f viral RNA containing a lethal 3Dpol mutation. RNA abundance measured by RT-qPCR. Data are means ± SD of N = 3 technical replicates. (D) circRNA IRES reporter transcript with split G/FP sequence flanking the IRES and upstream intron and downstream inverse repeat sequences (REP) driving backsplicing. (E) Immunoblots of GFP expressed by HAV, HCV, PV, EMCV, and KSHV circRNA IRES reporter plasmids transfected into sgCtrl or PDAP1-KO1.4 cells. (F) IRES-mediated translation efficiency in PDAP1-KO1.4 versus sgCtrl cells (100%), calculated as GFP/GAPDH abundance normalized to circRNA transcripts quantified by specific RT-qPCR. Data are means ± SEM from N = 3 to 6 independent transfections. *P < 0.05; **P < 0.01; ***P < 0.001, by one-sample t test and Wilcoxon test. (G) HAV RNA associated with polysomes in sgCtrl cells, PDAP1-KO1.4 cells, or PDAP1-KO1.4 cells transduced with lentivirus expressing PDAP1-Flag. Cells were harvested 5 hours after infection, and ribosomes were separated on a 10 to 50% sucrose gradient. Below are immunoblots of PDAP1-Flag and eIF4E in fractions from HAV-infected PDAP1-KO1.4 cells expressing PDAP1-Flag, with and without 50 mM EDTA. (H) Translation efficiency (percent RNA associated with polysomes) of HAV and β-actin mRNA in PDAP1-KO1.4 cells, relative to sgCtrl cells (100%) in two independent experiments. (I) [35S]-Met/Cys incorporated into trichloroacetic acid–precipitable material over 30-min incubation in PDAP1-KO1.4 cells normalized to sgCtrl cells (100%), with or without HAV infection. CHX, 100 μg/ml cyclohexamide (CHX). Data are means ± SD of N = 3 technical replicates. *P < 0.05; **P < 0.01; ***P < 0.001, by t test.
Fig 2: PDAP1 is an eIF4E-binding protein.(A) AlphaFold prediction of the PDAP1 structure showing the canonical 4E-binding motif and serine residues identified as sites of phosphorylation (AlphaFold Protein Structure Database Q13442) (32). aa, amino acid. (B) Amino acid alignment of PDAP1 with known eIF4E-binding proteins, including 4E-T and C8orf33. The YXXXXLΦ motif is boxed in red, with other conserved Lys/Arg residues boxed in blue. (C) Immunoblots of proteins immunoprecipitated (IP) by anti-Flag from lysates of cells expressing PDAP1-Flag or empty vector (“−”), with or without HAV infection. (D) Glutathione bead pull-down of bacterially expressed PDAP1 mixed with GST-eIF4E and GST-eIF4E coimmunoprecipitation with PDAP1. Purified recombinant proteins were mixed with a 300-fold molar excess of human serum albumin. (E) Immunoblots of proteins pulled down with glutathione beads from lysates of PDAP1-KO1.4 cells expressing PDAP1-Flag, PDAP1-Y124A-Flag, or empty vector (“−”) following addition of bacterially expressed GST-eIF4E. sgCtrl, control cells. (F) (Left) GFP expressed by the HAV circRNA IRES-GFP reporter in PDAP1-KO1.4 and control cells transfected with PDAP1-Flag, PDAP1-Y124A-Flag or empty vectors (“−”). (Right) Estimated IRES activity calculated as (GFP/GAPDH)/circRNA quantified by RT-PCR. P value by two-way ANOVA, N = 3 independent experiments. AU, arbitrary units. (G) 18f/NLuc reporter virus replication in PDAP1-KO1.4 cells reconstituted with wild-type or Y124A mutant PDAP1. EV, empty vector. (H) MS intensities of phosphorylated PDAP1 peptides identified by LC-MS following phospho-peptide enrichment of lysates from mock-infected or HAV-infected Huh-7.5 cells. Data are means of two technical replicates of each of three independent samples. Adjusted P value by ANOVA. (I) Impact of phospho-mimetic and phospho-ablative mutations on lentivirus-expressed PDAP1-Flag rescue of 18f-NLuc reporter virus replication in PDAP1-KO1.4 cells. P values by nonparametric Friedman test with Dunn’s correction for multiple comparisons.
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