Method details

Information about the plasmids, antibodies and reagents used in the study is given in key resources table.

The pP2A plasmid was derived from pcDNA3.1 containing an in frame FLAG epitope, the porcine teschovirus-1 2A peptide and EGFP (Chen et al., 2018) and used for construction of overexpression vectors. The pP2A-DDX3 (wild-type, S382LMe, K230E) vectors were previously described (Chen et al., 2015). OSCC-associated DDX3 mutations (D521H, D558H, E572Q) were generated from the pP2A-DDX3 (wild type) vector by using PCR-based mutageneis. The pP2A-AREG vector was constructed by inserting human AREG cDNA from SAS cells. In all of the DDX3 overexpression vectors, synonymous substitutions in the siD#1-targeting site were made to avoid knockdown in the compensation experiments. For transient knockdown of DDX3 partners, each of the gene-specific shRNA sequences was constructed into the pAAVEMBL-CB-EGFP vector as described (Chen et al., 2016). The AREG expression vector was constructed by cloning the human AREG cDNA 5’ in-frame with the FLAG tag into pP2A. To construct the in vivo translation reporters, the 5’ UTR (210 nt) and 3’ UTR (265 nt) sequences of AREG were respectively inserted upstream and downstream of the humanized renilla luciferase (hRL) gene of psiCHEK-2 (pCH, Promega.

Total RNA was extracted from control or DDX3-depleted SAS cells using Trizol Reagent according to the manufacturer’s instructions. Ribosomal RNA (rRNA) was depleted using the Ribo-off rRNA Depletion Kit. Library preparation was performed using the NETNext Ultra Directional RNA Library Prep Kit for Illumina according to the manufacturer’s instructions. The quality and quantity of the libraries were respectively analyzed by Fragment Analyzer Automated CE System (Advanced Analytical Technologies) and Qubit Fluorometer (ThermoFisher). Both RNA-seq and Ribo-seq (see below) were performed on the Illumina sequencing platform (NextSeq 500) following the manufacturer’s instructions, and assisted by Insight Genomics (Tainan, Taiwan).

The procedure of Ribo-seq was moderately modified from the protocol as described (Ingolia et al., 2012). Briefly, control or DDX3-depleted cells were lysed with the polysome buffer containing 20 mM Tris-HCl pH7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 200 μg/ml cycloheximide (CHX), 1% (v/v) Triton X-100 and 25 U/ml Turbo DNase. The lysates were subsequnetly digested with RNase I. After purification, rRNA was depleted using the Ribo-off rRNA Depetion Kit. Ribosome-protected fragments (RPF) were purified with PAGE and dephosphorylated with T4 Polynucleotide Kinase. Linkers were ligated to RPFs using T4 RNA ligase 2, truncated K227Q and the cDNA was synthesized using SuperScript III reverse transcriptase. The cDNA samples were circularized by CircLigase ssDNA Ligase, amplified by Phusion High-Fidelity DNA Polymerase and purified using AMPure XP for PCR Purification.

For analysis of the sequencing data, Cutadapt was used to remove the short adapter ‘CTGTAGGCACCATCAAT‘ from the RNAseq and Riboseq samples. Bowtie and TopHat2 were used to map the reads to the human genome version hg38/GRCh38.p13 with specific settings ‘--no-coverage-search --segment-length 14 --segment-mismatches 1’, and calculate the mapped reads statistically. The reads per kilobase pase million (RPKM) of each protein-coding gene for each sample, and the differential expressions of genes between various samples were calculated with Cufflinks and Cuffdiff 2 based on the protein coding gene annotation file provided from Ensembl database. After Cuffdiff normalization and elimination of the genes with no read coverage, the transcripts with siC RPKM higher than 1, p value lower than 0.05 and log2(siD/siC) larger than 1 and lower than 1 were selected for analysis.

Profiling of growth factors/cytokines in control or DDX3-depleted SAS cells was performed using Human Cytokine Antibody Array according to the manufacturer’s instructions. Briefly, cells were lysed in Cell Lysis Buffer. Protein concentration of the lysates was evaluated using the BCA Protein Assay Kit. For hybridization, 1 mg of the lysate proteins in 1 ml of the blocking buffer were incubated with array slides overnight at 4^°C. The slides were then sequentially incubated with Biotinylated Antibody Cocktail at 4°C overnight, and subsequently HRP-Conjugated Streptavidin Solution at 4°C overnight with thorough washing after each step. Images were captured using LAS-3000 Imager (Fujifilm), and the intensity of blots was analyzed using ImageJ.

Cell lysates were collected in the immunoprecipitation (IP) buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.5% Triton X-100, 5 mM MgCl₂) containing Protease/Phosphatase Inhibitor Cocktail. 400 ng/μl RNase A and 0.5 unit/μl RNasin were added for removing RNA-mediated interaction and RNA immunoprecipitation (RIP), respectively. For IP or RIP, 1 mg of cell lysate proteins were incubated with 3 μg of antibody that was bound onto Dynabeads Protein G in 1 ml of protease/phosphatase inhibitor-containing IP buffer at 4°C for 2 hrs. After washing with 1 ml of IP buffer for 5 times, beads were suspended in the sample buffer (125 mM Tris-HCl pH6.8, 11% glycerol, 2.4% SDS, 3% 2-mercaptoethanol, 0.01% Bromophenol Blue) or Trizol for immunoblotting or for RNA extraction, respectively. For mass spectrometry analysis of DDX3-interacting proteins, 5 mg of cell lysate proteins and 15 μg of DDX3 antibody were used. Protein bands separated by using SDS-PAGE were cut, digested by trypsin, purified and subjected for LC-ESI/MS/MS analysis. In the SILAC experiment, light and heavy isotope-labeled SAS cell lysates were immunoprecipitated without and with RNase A, respectively. After IP, samples were mixed, digested by trypsin, purified and subjected for 2D-LC-ESI/MS/MS. Ratios of heavy to light amplitude of proteins higher than 2 were picked to represent RNA-independent interacting proteins of DDX3.

The AREG level of culture medium was measured by using the AREG ELISA Kit according to manufacturer’s instructions. Briefly, 100-μl DMEM or CM was added to each well of the AREG microplate and incubated at 37°C for 1 hr. After discarding the medium, wells were incubated with 100 μl of 1× Biotinylated AREG Detector Antibody at 37°C for 1 hr. After thorough wash with 1× Wash Buffer, wells were incubated with 100 μl of 1× Avidin-HRP Conjugated at 37°C for 30 min. After thorough wash with 1× Wash Buffer, wells were incubated with 90 μl of TMB Substrate at 37°C in the dark for 20 min. Finally, 50 μl of Stop solution was added, and optical absorbance at 450 nm was measured immediately by using SpectraMax 190 Microplate Reader (Molecular Devices).

Total RNAs were extracted using the HiYield Total RNA Extraction Kit and then subjected to DNase digestion on beads according to manufacturer's instructions. RNAs were reversely transcribed using the ToolsQuant II Fast RT Kit according to manufacturer’s instructions. cDNAs was analyzed by qPCR using PerfeCta SYBR Green FastMix PCR Reagent in LightCycler480 instrument (Roche).

Immunofluorescence staining was performed as previously described (Chen et al., 2012). Confocal imaging with a Plan-Apochromat 100x/1.4 Oil objective was carried out by using ZEISS LSM with Airyscan super-resolution mode. Histograms of fluorescence intensities were produced by using ImageJ.

For knockdown experiments, SAS cells were transfected with siRNA or shRNA 48 hours prior to reporter transfection. For reporter transfection, 0.05 μg of pCH or pCH-AREG UTR reporters and 0.45 μg of pP2A or DDX3 overexpression vectors were used. Luciferase assay was performed by using the Dual-Luciferase Reporter Assay System 24 hours after reporter transfection. The activity of humanized firefly luciferase encoding by the same pCH reporters was used for references. The same cell lysates were used for RT-qPCR and immnunoblotting analysis.

Fractionation of the cytosol and membrane fractions was performed as described previously (Jagannathan et al., 2011) with minor modification. Briefly, SAS cells with 80–90% confluent in a 10-cm plate were washed with PBS, treated with PBS containing 50 μg/ml CHX and then incubated with 0.5 ml of permeabilization buffer (110 mM KOAc, 25 mM K-HEPES pH7.2, 2.5 mM Mg(OAc)₂, 1 mM EGTA, 0.3% digitonin, 1 mM DTT, Protease/Phosphatase Inhibitor Cocktail, 40 U/ml RNasin, 50 μg/ml CHX) for 5 min to collect cytosol lysate. Cells were then washed with wash buffer (110 mM KOAc, 25 mM K-HEPES pH7.2, 2.5 mM Mg(OAc)₂, 1 mM EGTA, 0.004% digitonin, 1 mM DTT), and then incubated with 0.5 ml of lysis buffer (400 mM KOAc, 25 mM K-HEPES pH7.2, 15 mM Mg(OAc)₂, 1% NP-40, 0.5% sodium deoxycholate, 1 mM DTT, Protease/Phosphatase Inhibitor Cocktail, 40 U/ml RNasin, 50 μg/ml CHX) for 5 min to collect membrane lysate. Both lysates were centrifuged at 7500×g for 10 min and supernatants were collected as the cytosol and membrane fractions. To perform polysome fractionation, 0.4 ml of the samples were overlayed on the 15–40% sucrose gradient and centrifuged at 38,000 rpm for 2 hrs. Polysome fractions were collected and monitored via an automatic gradient fractionator (ISCO). Nine fractions from 40S to the first polysome and the following nine fractions were collected as the light and heavy fractions, respectively. Trizol LS reagent was used for RNA extraction from the fractions.

Biotin-labeled AREG 3′ UTR RNA was synthesized by using T7 RNA polymerase according to the manufacturer's instructions except that 3.2 mM MgCl₂ and 1 mM Bio-16-UTP were additionally added. RNA was recovered by using Trizol reagent and the quality of RNA was checked by electrophoresis. Cell lysates collected in the pulldown buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl₂ and 0.1% NP-40) supplemented with Protease/Phosphatase Inhibitor Cocktail and 40 U/ml RNasin. Pulldown was performed in a final volume of 200 μl containing 10 μl of Dynabeads MyOne Streptavidin C1 and 1 mg of lysate with or without 0.5 μg of biotin-labeled AREG 3′ UTR RNA at room temperature for 4 hrs. After thorough wash with IP buffer, immunoblotting was performed to reveal RNA-associated proteins.