Transcription factor Wilms’ tumor 1 regulates developmental RNAs through 3′ UTR interaction

Bharathavikru et al. show that Wilms’ tumour 1 (WT1) binds preferentially to 3′ UTRs of developmental targets, which are down-regulated upon WT1 depletion in cell culture and developing kidney mesenchyme. Combining experimental and computational analyses, they propose that WT1 influences key developmental and disease processes in part through regulating mRNA turnover.

2 the samples were subjected to phenol-chloroform extraction and RNA precipitation with glycogen, Sodium acetate and absolute ethanol. The precipitated RNA was quantified by nanodrop and agilent bioanalyzer. Samples were processed either for next generation sequencing or qRT-PCR.
Next-generation sequencing: Double stranded cDNA synthesis of RNA-IP samples was performed as follows, the first strand cDNA synthesis was initiated using random primers, incubated at 70ºC for 10 minutes, followed by incubation on ice and reverse transcriptase reaction using AMV RT at 42ºC for 60 minutes, incubated on ice to terminate the reaction.
This was followed by the second strand synthesis using the second strand enzyme and incubated at 16ºC for 2 hours, 10 µl of T4 DNA polymerase was added and incubated at 16ºC for 5 mins. The above reaction was terminated by the addition of 8.5µl of 0.2M EDTA, pH 8.0. Samples were then subjected to RNA digestion using RNase and proteinase K digestion, both at 37 ºC for 30 mins each followed by cleaning of the dsDNA product by phenolchloroform extraction and precipitation with 5M ammonium acetate and 2.5 volumes of absolute ethanol. The samples were tested on bioanalyzer before being processed by the NEB next sequencing kit for illumina for NGS on Illumina platform. MgCl2, 1 mM DTT, protease inhibitors (Roche, complete, EDTA-free), RNAse Inhibitor (Promega)), followed by addition of 10u of RQ1DNAse (Promega) to the samples and mixed by pipetting followed by incubation at room temperature for 10 min. Lysates were centrifuged at 14000rpm for 10 min at 4ºC and supernatant was collected.
Antibody preparation: Antibodies used were IgG conjugated protein A agarose beads and agarose beads conjugated with WT1 C19. The antibodies were washed with PBS/0.4% NP40 and incubated with the cell lysates for 60 min at 4°C. Supernatant was discarded, followed by beads washed twice with PBS-WB buffer (PBS reconstituted with 150mM NaCl, 2 mM MgCl2, 0.4% NP-40), and once in 1xPBS buffer with 2mM MgCl2.
Since this is an endogenous protein IP, formaldehyde crosslinking was done on the RNP complex bound beads using 0.5% formaldehyde in PBS for 3 min, followed by addition of 0.2 M glycine and 0.1 M Tris-HCl pH 8 and incubated for 5 min. Crosslinked complexes were subjected to denaturing washes (4 times) in urea buffer (20 mM Tris pH 7.4, 8 M UREA, 0.3 M NaCl, 0.4% NP-40) and additional incubation in urea buffer for 30 min at 4ºC.
Linkers' ligation and RNA-protein complexes recovery: WT1 RNP associated beads were subjected to washes with PNK buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.5% NP-40, 50 mM NaCl), four times. To remove unwanted 3'phosphate groups from bound RNA fragments the complexes were treated with TSAP phosphatase (Promega) using provided buffer for 40 min at room temperature. To inactivate the enzyme, the beads were washed twice with UB and four times with PNK buffer. This was followed by the 5' phosphorylation and radioactive labelling of RNA. The complexes on the beads were incubated with 40 units of T4 Polynucleotide kinase (New England Biolabs), initially with P 32 labelled ATP for 45 min, followed by 1 mM cold ATP for 20 min, in PNK buffer with RNase inhibitors (RNasin, Promega) at room temperature. The reaction should provide 5' phosphates needed for downstream ligations. The beads were then washed as before, twice with urea buffer and four times with PNK buffer. WT1-bound RNA molecules were ligated together and with 3' linker (1 μM miRCat-33, IDT), overnight using 40 units of T4 RNA ligase 1 (New England Biolabs) in PNK buffer with RNase inhibitors at 16°C followed by washes as the previous step. Then using 40 units of RNA ligase 1, barcoded 5′ linkers (final conc. 5 μM; IDT, one for each sample) were ligated in RNA ligase 1 buffer with 1mM ATP for 3-6 hr at 20°C. The beads were washed as before with urea buffer and PNK buffer. Samples were boiled in NuPAGE protein sample buffer containing 100 mM Tris-HCl, 1%SDS, 100 mM βmercaptoethanol for 3 min. The samples were cooled on ice for 5 min and centrifuged to recover the supernatant with RNA-protein complexes. 10% methanol for 1 hr at 100V. The membrane was exposed on film (Amersham) for 1 hr or overnight at -70ºC depending on the strength of the signal. The film was developed and aligned with the membrane to facilitate the isolation of radioactive bands corresponding to the WT1-RNA complexes.
cDNA Library preparation: The isolated RNA was dissolved in 12 µl of distilled RNAsefree water and reverse transcribed using miRCat-33 primer (IDT) with Superscript III Reverse Transcriptase (Life Technologies) for 1h at 50°C, followed by RNase H (New England Biolabs) treatment for 30 min at 37°C. cDNA was amplified using primers P5 and primer PE miRCat_PCR and TaKaRa LA Taq polymerase (Takara Bio). PCR products were separated on a 2% MetaPhor agarose (Lonza) gel with SYBRSafe (Life Technologies) in 1 x TBE at 4ºC. The gel band corresponding to 150-200bp was excised and the cDNA from this band was purified with MinElute Gel Extraction Kit (QIAGEN). The purified cDNA libraries were sequenced by NGS on Illumina platform.

qRT-PCR validation:
Total RNA was isolated as described in the transcriptome analysis for gene expression analysis. Alternatively, for RNA interaction experiment validations, interacting RNA was precipitated as explained in the respective sections. The precipitated RNA was processed for cDNA synthesis instead of library preparation. cDNA synthesis was done by using the promega GOTaq qRT system using either oligodT or random primers for reverse transcription based on the experimental requirement. Most of the gene expression analysis studies were done using oligodT primers whereas the IP validations were carried out using the random primers. SyBR Green was used for detection. 18s rRNA was used as the control. Gene expression data was analysed by the del del Ct method. ES cells were compared to KO ES cells. For M15, since there was a 0.5 fold reduction of Wt1 levels in lacZ knockdown in relation to the M15 control (Supplemental Fig. S4), for all calculations, lacZ and Wt1 knockdown were compared. The IP data was analysed using the fold enrichment method. Students' unpaired t-test was used for statistical validations.

Motif Analysis:
Integrating the kmer analysis with the transcriptome changes, the top 10 kmers (ranked by Z score) located in 3' UTRs were found to be associated with 892 genes, 19 of which were downregulated in ES whereas none in the upregulated category. In M15, the top 10 3' kmers occur in 1229 genes, 24 of which are downregulated and none upregulated.
This sequence is similar to the PUM2 RNA binding motif TGTANATA (Hafner et al. 2010).
These findings indicate that WT1 binds in the vicinity of the Poly (A) site and may either bind near Pum2 or have a comparable miRNA interaction mechanism to that proposed earlier functional analysis using the GO FAT category, was used.
Immunoprecipitation of endogenous WT1: Approximately 1x10 6 WT1 expressing cells were lysed in RIPA buffer (50mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X 100, 0.5% Sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail) and the protein extract was quantified. The lysate was pre-cleared using protein A/G agarose beads for 1 hour at 4ºC. About 0.25 mg of the lysate was used per IP reaction with 2µg of antibody (agarose bound WT1  or agarose bound IgG (SC2345)). After overnight IP at 4ºC, the beads were washed with RIPA buffer (1ml x 4 times) and PBS (1 x 2 times). After the final PBS wash, the beads were resuspended in SDS loading buffer and processed for western blotting analysis. Most primary antibodies were left overnight at 4ºC at a dilution of 1:2500 and the secondary antibody was used at 1:10000 dilution for an hour at room temperature. The results were visualized with the help of the thermo scientific ECL kit. Antibodies used were as follows: WT1 (C-19, SC192/X), secondary antibody conformation specific anti rabbit HRP conjugate (CST L27A9).

RNA stability experiments: 5x10 4 Mouse ES and Wt1 knockout ES cells as well as the M15
lacZ knockdown control and Wt1 knockdown cells in 24 well plates were treated with actinomycin at 10µg/ml concentration. Cells were collected after 0, 3, 6 and 8 hours of treatment, resuspended in Trizol (100 µl), followed by addition of chloroform (20 µl) and vortexed. Samples were centrifuged at 13000 rpm for 15 minutes at 4ºC. Supernatant transferred to new 1.5ml tubes with 1µl of genelute. RNA was precipitated by addition of ethanol (70 µl), samples were vortexed and incubated at room temperature for 10 minutes, followed by centrifugation at 13000 rpm for 10 minutes at 4ºC. RNA pellets were washed with 70% ethanol and resuspended in 15 µl of RNase free water. RNA concentration was estimated by nanodrop and following oligodT primed cDNA synthesis, qRT-PCR was done as explained earlier using βactin as the reference gene.
Luciferase Reporter Assay: WT1 interacting hybrid sequences were separated to each half of the hybrid. This sequence was confirmed to be present both in the WT1 interacting single reads from FLASH dataset as well as the RIP-seq dataset. Primers were designed so as to include about 100bp flanking sequence to the WT1 interacting region referred to hereafter as binding site (BS). Each half of a hybrid thus amplified was denoted as BS1 and BS2. PCR amplified fragments were cloned into the pIS1 vector (Hmga2 3'UTR wt luciferase (Luc-wt) was a gift from David Bartel (Addgene plasmid # 14785) using the XbaI-NotI sites thus replacing the HMGA2 fragment with the binding sites, which were confirmed by restriction digestion confirmation and sequencing. pIS0 plasmid was used as the luciferase transfection control. 1x10 5 M15 lacz control knockdown and Wt1 knockdown cells were transfected with 1µg each of the pIS0 transfection control plasmid and different versions of the binding site pIS1 based plasmids as indicated using 4 µg of lipofectamine. Following overnight transfection, media was changed, 30 hours post transfection, cells were collected and lysed with passive lysis buffer (500 µl). Luciferase measurements were done on a fluostar omega plate reader using the Dual Luciferase Reporter Assay reagents as per manufacturer's instructions. Briefly, 50 µl of the LAR II reagent was pipetted into wells. 20 µl of the lysate was added to these wells, mixed by pipetting and firefly luciferase (control) measurements were recorded. This was followed by addition of 50 µl of the Stop and Glo reagent, mixed by pipetting, followed by measurements of renilla luciferase (with the WT1 binding sites).
Measurements were done on biological duplicates and technical triplicates. Background luminescence was detected based on measurements from the untransfected controls. Renilla activity to Firefly activity ratios were obtained as normalized to transfection efficiency.

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Control vector with no binding site was used as a reference to compare the activity with the PBS1 (Podxl binding site 1) and IBS1/ IBS2 (Igfbp5 binding site 1/2). GFP Reporter Assay: Igfbp5 binding site 2 (IBS2) as explained above was PCR amplified and cloned into EcoRI/NotI sites of GFPd2 plasmid (pCAG-GFPd2 was a gift from Connie Cepko (Addgene plasmid # 14760) in tandem with the UTR, and confirmed by restriction digestion and sequencing. 1x10 5 M15 lacz control knockdown and Wt1 knockdown cells were transfected with 1µg of either GFPd2 vector alone or the IBS2-GFPd2 plasmid using 2.5 µg of lipofectamine. Following overnight transfection, media was changed. Cells were collected after 0, 3, 6 hours post media change and lysed with RIPA lysis buffer (0.1 ml). Protein concentrations were estimated and 20 µg whole cell lysates were electrophoresed on 10 % SDS PAGE. GFP reporter expression was assessed by immunoblotting with GFP antibody (TA150041). WT1 expression was confirmed with antibody against WT1 (ab 89901) and HSP90 antibody (BD biosciences, 610419) was used to assess equal loading.

B) Motifs identified by k-mer analysis on targets identified in ES RIP-seq and M15 RIP-seq
analysis. The MEME motif identified is also presented which shows a GC rich recognition motif.
C) The top 10 recurring kmers in 3' UTRs identified in WT1 FLASH single read data, ordered by Z score (top). Kmers reordered by enrichment for downregulated genes (bottom, ** p<0.01 * p<0.05). Table showing Tables   Table S1: Gene ontology terms identified by GOrilla analysis of top 1000 WT1 interacting mRNA, identified in the ES cell line, WT1 RIP-seq dataset.   and WT1 RIP-seq in M15 cells. Sheet 1 provides a summary, Sheet 2 is the FLASH dataset analysis, Sheet 3 is the WT1 RIP-seq data from ES cells and Sheet 4 is the WT1 RIP-seq data from M15 cells. Table S5: List of miRNA binding sites identified by TargetScan in the WT1 interacting RNA that form intramolecular hybrids at the 3' UTR. Sheet 1 is a list of target site and the number of hybrids with a defined miRNA binding site. Sheet 2 is the sequence information of the above. Sheet 3 represents the different miRNAs that can target these hybrids.