Supporting Information
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Supporting Information SI Methods EWS-FLI1 interacting partners identified through MS. We developed an unbiased screen for potential partners that interact with EWS-FLI1 by adding ES nuclear lysate to immobilized EWS-FLI1, followed by elution and PAGE. Nuclear lysate from Ewing sarcoma (ES) cell line TC32 was added to two Hi-TRAP Ni charged columns in series, with only the second column containing EWS-FLI1. Following the addition of nuclear lysate and wash, the columns were separated and independently eluted with imidazole. Fractions were resolved on both high (14%) and low (6%) polyacrylamide gels, followed by Coomassie staining and protein bands of interest were manually excised and subjected to in-gel tryptic digestion. Protein digests separation was performed on an Aquity UPLC BEH 130 C18 column and followed by mass spectrometry (MS). Mass spectra were acquired online using a nano-ESI-LC-MS/MS QStar Elite (ABSCIEX) and operated with Analyst QS v2.0 data acquisition software. MS data were searched against the SWISS-PROT databases using ProteinPilot software 3.0 and Paragon Algorithm to identify the individual proteins. Positive identification of proteins eluting with EWS-FLI1 was rigorous including: (i) band from the gel located near estimated molecular weight, (ii) greater than 95% ion confidence interval, and (iii) 99% confidence in peptide assignment based upon greater than 3 peptides. Proteins eluted from a Ni-charged column without EWS-FLI1 were considered non- specific. Proteins were identified in three separate experiments starting from EWS-FLI1 and fresh nuclear lysate to insure reproducibility. Nuclear Co-Immunoprecipitation. Co-Immunoprecipitation (Co-IP) was used to pull down protein partners of a protein of interest using an antibody that specifically binds to this specific protein in order to test protein-protein interaction. Active motif universal magnetic Co-IP kit (Active Motif) was used to make nuclear extract from TC32 cells. The protein G magnetic beads were used for Co-IP and the IP was performed on 300 μg samples using 2 μg of FLI1/PRPF8/DDX5 polyclonal antibody and rabbit IgG (as a negative control). Western blot was then performed individually using the following antibodies FLI1 (Santa Cruz Biotechnology, USA), DDX5, PRPF6, hnRNP K, RHA, hnRNP U, CDC5L, DDX17, SF3B3, SF3B2, SF3A2, PRPF8, SFPQ (Abcam, USA) and snRNP200 (Sigma-Aldrich, USA), on 10 μg input nuclear extract, FLI1/PRPF8/DDX5 IP and the rabbit IgG IP. ELISA. ELISA was performed on maxi sorb 96 well plate. Recombinant full length EWS-FLI1 protein (300 ng) were coated on the bottom surface of the ELISA plate overnight and followed by blocked with 4% BSA. Spliceosomal proteins DDX5, PRPF6, hnRNP K, RHA, hnRNP U, CDC5L, DDX17, SF3B3 and SF3B2 were used as ligand to detect the specific protein interaction using anti-DDK monoclonal antibody with HRP conjugated (for RHA protein goat anti-RHA antibody (Everest)), protein binding was detected using 3,3’,5,5’-tetramethylbenzidine (TMB) peroxidase substrate kit (Bio-Rad, CA) per the manufacturer’s instructions. The substrate reaction is incubated at room temperature with periodic mixing (e.g., 2-3 seconds shaking in the plate reader) and the color development monitored by absorbance reading at 415 nm or 405 nm using a microplate reader. Negative controls for each ELISA included the absence of EWS-FLI1, absence of second recombinant protein, and blocking alone were used. For the positive control, ELISA plate wells were coated with their corresponding spliceosomal proteins and their corresponding secondary antibody. 1 Method for drawing networks. The proteins identified by MS as a potential interactive partner of EWS-FLI1 are included and followed by proteomic analysis were used as input for Ingenuity Pathway Analysis to identify networks of interacting proteins. Using IPA, clusters of proteins appearing in the top functional pathways and networks with the most highly significant p-values were selected for constructing the network. IPA uses a curated proprietary knowledgebase to identify protein interactions based on experimental evidence gathered from published reports. The resulting networks thus generated were filtered for direct and indirect interactions, and trimmed to remove nodes beyond the second level of interactions from the proteins of interest. Direct binding partners that appeared within this network were confirmed by experimental validations. Affymetrix Exon Array. Total RNA was extracted using Qiagen AllPrep DNA/RNA/Protein kit from each of the derived cell lines (9 cell lines with/without EWS-FLI1). The RNA quality was measured using Agilent 2100 bioanalyzer and RNA Integrity number (RIN) above 9.60 were used for Exon array experiments. cDNA was synthesized and hybridized to Affymetrix GeneChip Human Exon 1.0 ST microarray chips and .CEL files are generated. These raw data files were processed using two analytical tools, PGS and easyExon. All data CEL files were uploaded to GEO and their Accession number is GSE65941. Data preparation and analysis for PGS. Data was pre-processed using Partek Genomic Suite software (PGS v6.6, Partek Inc., St. Louis, MO) with the Affymetrix-annotated core probe set. The configuration consisted of a pre-background adjustment for GC content and probe sequence, Robust Multi-array Analysis (RMA) (1) for background correction, quantile normalization and probe set summarization using median polish. All signals were log2-transformed. Library files were those specified by Affymetrix (hg18). Differential expression was determined by ANOVA. The multiple test correction implemented the p-value method to determine a false discovery rate (FDR) < 0.05. Exon level data was filtered using Detection Above Background (DABG; (2)). Data was analyzed for alternative splicing by a repeated measure AS-ANOVA. The p-value method was used to control the FDR with a cutoff <0.05 to determine the presence of alternative exon usage. Data analysis for easyExon. easyExon uses RMA for background correction and probe set summarization. Microarray detection of alternative splicing (MIDAS) p-value <0.05 and pattern- based correlation (PAC) are used to determine significant AS transcripts (3). The degree of AS was visualized by the spicing index (SI) plot. In addition, the intensity fold change > ±2 was also calculated to show the differentially expressed genes. Exon array validation by Quantitative real-time polymerase chain reaction (qRT-PCR). Exon array findings (both expression and alternative splicing) were validated using qRT-PCR on the several genes focused on in this study. Validation of alternative splicing was performed using 2 different primer-pair combinations within the same transcript (RefSeq - release 61). One pair of primers target an area that can be used to differentiate between isoforms, and a second pair target an area common to all transcripts of the gene. The latter was used to normalize endogenous expression. Transcript expression validation used 18S as the internal normalizer gene with the primer set for the transcript of interest. 2 RNAseq analysis and MISO. 100 million paired-end reads were aligned to hg19 using TopHat2/Bowtie2 (4, 5) with default parameters for genome-guided transcriptome reconstruction. MISO version 0.5.2 was used to estimate PSI values (6). Version 2 reference annotation includes annotated splicing events found in Ensembl genes, UCSC knownGenes, and RefSeq genes. Version 2 includes annotation of skipped exons, retained introns, mutually exclusive exons, and alternative 5’/3’ splice sites. Discovered splicing events were filtered for those with a PSI difference greater than 20% as well as a Bayes factor greater than 10. Probability of overlap between sets of splicing events found in either shEWS-FLI1 or YK-4-279 was calculated using a hypergeometric distribution specific to the total number of annotated events of each type. RNA immunoprecipitation (RIP) and Cross-linked RNA immunoprecipitation (CLIP). RIP was carried out as previously published (7). CLIP was carried out using FLI1 antibody from Innovagen (Sweden) raised against recombinant FLI1 C- terminal protein in rabbit. TC32 cells were UV crosslinked at 150 mJ/cm2 in Stratalinker (Invitrogen). The CLIP protocol was followed as published previously (8). CLIPseq analysis and MEME suite. CLIPseq reads were trimmed for barcode and CLIP adapter sequences and PCR duplicated were removed. Filtered reads were then aligned to hg19 using Bowtie version 1.1.1 (9, 10). Those reads that aligned unambiguously were used in downstream processing. Sequence of forward and reverse reads were treated separately as MEME inputs (11). The top motif with a corresponding reverse complement motif in the reverse set was considered as the lead motif for further analysis. Motif localization and secondary structure MFE prediction. All higher-level data analysis was done in R using Bioconductor packages or at the UNIX command line (12, 13). The BSgenome and GenomicRanges R packages were used to extract genomic sequence +/-125bp of the 5’ and 3’ ends of all exons annotated in hg19. These sets were searched for occurrences of the lead motif with ≥ 90% match to the motif position weight matrix. Frequency of hits were graphed with respect to the exon-intron boundary for the 5’ and 3’ sets separately. Local secondary structure prediction was performed using RNALFold version 2.1.8. Local secondary structure was defined as 200nt or less, not including g-quadruplex formation. Minimum free energy for each read sequenced and aligned greater than 20nt long was recorded. A null set was generated by taking random exonic sequences of the same length as each CLIP read. These two minimum free energy density profiles were plotted together to compare local secondary structure preference of EWS-FLI1 RNA binding. Minigene cloning and transfection. TERT minigene was synthesized using the three exons and two introns (300 bp on the either side of exon-intron junction) and subcloned them into pcDNA3.1+ vector (GenScript). We transfected a total of 6 μg of minigene plasmid into 50% confluent TC32 cells using X-treme Gene 9 DNA transfection reagent (Roche Diagnostics) (14).