Skip to main content
NCBI home page
RNA Biology logoLink to RNA Biology
. 2015 Oct 29;12(12):1364–1371. doi: 10.1080/15476286.2015.1102831

HuR antagonizes the effect of an intronic pyrimidine-rich sequence in regulating WT1 +/−KTS isoforms

Hui Li 1,#, Shuai Hou 1,#, Tian Hao 1, Sikandar Azam 1, Caigang Liu 2, Lei Shi 1, Haixin Lei 1,*
PMCID: PMC4829285  PMID: 26512748

Abstract

WT1 + KTS and −KTS isoforms only differ in 3 amino acids in protein sequence but show significant functional difference. The +/−KTS isoforms were generated by alternative usage of 2 adjacent 5’ splice sites at RNA level, however, how these 2 isoforms are regulated is still elusive. Here we report the identification of an intronic pyrimidine-rich sequence that is critical for the ratio of +/−KTS isoforms, deletion or partial replacement of the sequence led to full/significant shift to -KTS isoform. To identify trans-factors that can regulate +/−KTS isoforms via the binding to the element, we performed RNP assembly using in vitro transcribed RNA with or without the pyrimidine-rich sequence. Mass spectrometry analysis of purified RNPs showed that the element associated with many splicing factors. Co-transfection of these factors with WT1 reporter revealed that HuR promoted the production of −KTS isoform at the reporter level. RNA immuno-precipitation experiment indicated that HuR interacted with the pyrimidine-rich element in WT1 intron 9. We further presented evidence that transient or stable over-expression of HuR led to enhanced expression of endogenous −KTS isoform. Moreover, knockdown of HuR resulted in decreased expression of endogenous −KTS isoform in 293T, SW620, SNU-387 and AGS cell lines. Together, these data indicate that HuR binds to the pyrimidine-rich sequence and antagonize its effect in regulating WT1 +/−KTS isoforms.

Keywords: alternative splicing, HuR, RNP purification, WT1, +/−KTS isoforms

Introduction

More than 90% of human coding genes undergo alternative splicing in specific tissues or developmental stages,1 whereas alternative splicing is a major determinant for the diversity of proteins in higher eukaryotes.2 Due to alternative splicing and usage of different translation starting codons, many variants have been detected for Wilms’ tumor gene 1 (WT1), one of the first cloned tumor suppressor genes.3 WT1 locates at chromosome 11p13 and contains 10 exons. There are 2 major alternative splicing events in WT1 pre-mRNA, the first one refers to inclusion or skipping of exon 5, and the second one involves alternative usage of 2 adjacent 5’ splice sites at the end of exon 9 which results in insertion or removal of 3 amino acids Lys-Thr-Ser (KTS) in between of zinc fingers 3 and 4, these 2 isoforms have been designated as +KTS and −KTS isoforms.4

WT1 has a versatile role in various biological and pathological processes, including development,5 epigenetics,6 apoptosis7 and tumorigenesis.8 Germline mutations of WT1 not only cause Wilms’ tumor, a pediatric tumor of the kidney,9,10 but also lead to urogenital malformations.9,11 During earlier stage of nephrogenesis, lack of WT1 induces apoptosis of the metanephric mesenchyme and renal abnormalities, while in later kidney development, WT1 impairs kidney cells proliferation and differentiation.12 The role of WT1 during hematopoiesis and leukemogenesis is similar to that observed in nephrogenesis. During early myeloid differentiation, WT1 expression is upregulated, while at later stages of differentiation its expression is downregulated.13 WT1 was first discovered as a tumor suppressor in Wilms’ tumor. However, latest research revealed that WT1 may act as an oncogene.14 Actually, WT1 may function as oncogenic or tumor suppressive gene, depending on cell types, differentiation status, microenvironment and presence of other gene alterations.15

WT1 +KTS/−KTS isoforms only differ in 3 amino acids in protein sequence, but show striking distinct functions in addition to many functional similarities. While +KTS and −KTS isoforms show similar function in the development of heart, spleen, and adrenal gland, they have distinct functions in renal development and sex-differentiation. For example, lack of +KTS causes the Frasier syndrome as well as defects in nepheron’s function. In contrast, absence of −KTS isoform leads to abnormal and hypoplastic kidneys.16 Furthermore, compared with −KTS isoform, +KTS isoform plays a crucial role in male-sex-determination. The insertion of KTS results in poor binding to DNA for +KTS isoform compared to −KTS isoform. The two main isoforms of WT1 (+KTS and −KTS) reside in different nuclear compartments.17 The +KTS isoform localizes with small ribonucleoproteins (snRNPs) and splicing factors within 30-50 nuclear speckles, being involved in posttranscriptional processes.18 The −KTS is usually found throughout the nuclear regions.12 The −KTS acts as a transcription factor and has been found to be active in transcriptional regulation by its strong tendency for binding DNA.18 Genes regulated by −KTS transcriptionally include PDGFA, WT1, EGFR, IGF2, and PAX2 that were expressed during kidney development.17,19-22 The +KTS interacts with the splicing factor U2AF65 (U2 small RNA auxiliary factor65) and regulates gene expression post-transcriptionally through binding with RNA.23,24 Further study reveals that +KTS/−KTS isoforms also have different role in tumorigenesis, WT1 −KTS isoform functions as tumor suppressor in mammary epithelial cells, it induces the up-regulation of p21, slows down cell proliferation and blocks cell cycle in G2 phase. In comparison, +KTS isoform functions to promote tumorigenesis by inducing EMT concomitant with expression variation of E-caderin and vimentin without apparent impact on p21 expression or cell proliferation.25

In contrast to the continuing expanded understanding in the functions of WT1 +KTS/−KTS isoforms, how the ratio of these 2 isoforms is regulated is still elusive. The generation of +KTS/−KTS isoforms is determined by the usage of 2 alternative 5’ splice sites, one of the 5 major categories of alternative splicing. In general, alternative splicing is regulated by trans-acting splicing factors recruited via the binding to cis-acting splicing regulatory elements (SREs).26-27 Depending on the location of the cis-elements and their final effects, SREs can be categorized into exonic or intronic splicing enhancers or silencers (ESEs, ESSs, ISEs and ISSs).28-29

Previous studies indicate that splicing enhancers bind the serine-arginine family of proteins (SR proteins) for their function, while splicing silencers recruit heterogeneous nuclear ribonucleoproteins (hnRNPs) to activate or suppress splice site recognition.30-32

In this study, we show that a pyrimidine-rich sequence in intron 9 close to the 5′ splice sites serves as a SRE favoring the production of the +KTS isoform. We further show that this sequence associates with many splicing factors including HuR, also known as ELAVL1. Overexpression of HuR promotes the production of −KTS isoform at the reporter level as well as the endogenous level, while depletion of HuR decreases the proportion of the −KTS isoform. Together, our data indicate HuR binds to the pyrimidine-rich sequence and antagonizes its effect in regulation of +/−KTS isoforms.

Results

Endogenous expression of WT1 +/−KTS isoforms

To determine the endogenous expression of WT1 +/−KTS isoforms, cDNAs from HeLa and 293T cell lines were amplified using WT1 specific cDNA primers localizing in exon 9 and 10. β-actin PCR products amplified from the same samples were used as internal control. As showed in Fig. 1A, WT1 expression was high in 293T cells but barely detectable in HeLa cells. Separation of the PCR products on polyacrylamide gel showed 77% of +KTS vs 23% of −KTS isoform (Fig. 1A). These results indicated that +KTS isoform was the dominant isoform expressed in 293T cells.

Figure 1.

Figure 1.

Open in a new tab

A pyrimidine-rich sequence in WT1 intron 9 regulates the ratio of +/−KTS isoforms. (A). Endogenous expression of WT1 +/−KTS isoforms in HeLa and 293T cells. (B). Schematic of WT1 reporter construct. Exons are shown as black boxes and introns as blue line. Alternative spliced sequence is shown as red box, and the pyrimidine-rich sequence is shown as yellow box. Intron 9 were truncated as indicated by double slashes. Exon sequence is shown in capital letters and intron sequence is shown in small letters. Deleted or replaced sequences are indicated, short sequences on top of the intron sequence are used to replace the indicated pyrimidine-rich sequences. Tn, T1, T2 and T3: pyrimidine-rich sequences; d: deleted; r: replaced. (C). Proportion of -KTS isoform from wild type WT1 reporter or mutant reporters after transient transfection to HeLa cells. SD: standard deviation. Percentage and SD are calculated based on 3 independent experiments. (D). Sequencing chromatography of -KTS and +KTS isoforms. Nine extra nucleotides in +KTS isoform are underlined.

A pyrimidine-rich sequence in intron 9 is critical for +KTS isoform

To see whether the +/−KTS isoforms can be generated using minigene, we created WT1-E9/10 reporter gene (Fig. 1B). Transient transfection of this reporter to HeLa cells results in 2 isoforms as detected by RT-PCR (Fig. 1C), the identity of which was further verified by sequencing after the bands were cloned into T vector (Fig. 1D). The production of +/−KTS isoform is due to the selection of 2 adjacent 5’ splice sites, we deleted a 32nt pyrimidine-rich sequence in intron 9 to test whether this sequence could regulate 5’ splice site selection. Strikingly, this deletion resulted in a complete shift to −KTS isoform (from 29% to 93%). Similarly, partial replacement of the pyrimidine-rich sequence (T1 or T2) led to significant increase of −KTS isoform (82% and 75%, respectively), whereas replacement of T3 showed a mild increase of −KTS isoform (39%). In addition, replacement of T1/T2 or T2/T3 resulted in higher proportion of −KTS isoform (94% and 85%, respectively) compared to single replacements. Together, these data indicated that the pyrimidine-rich sequence played a critical role in the production of +KTS isoforms.

The pyrimidine-rich sequence associates with pre-mRNA splicing factors

We next sought to identify proteins that bind specifically to the pyrimidine-rich sequence. To do this, we generated E-Tn construct containing part of WT1 exon 9 and intron 9 and E-dTn construct with the pyrimidine sequence deleted (Fig. 2A). RNP assembly was carried out on in vitro transcribed RNAs labeled with biotin and 32P, the mixtures were loaded on Sephacryl S-500 column and the chromatography was showed in Fig. 2B. The RNPs were then isolated using streptavidin agarose resin and an aliquot was separated on polyacrylamide gel (Fig. 2C). Total proteins were analyzed by mass spectrometry after TCA precipitation. RNAs only labeled with 32P but not with biotin were included as negative controls. Both RNPs contained a large number of RNA binding proteins including many hnRNPs (Table 1 and Suppl. Table S1), whereas a group of splicing related factors including SRSF3, SRSF6, SRSF9, TRA2A, TRA2B, HuR, hnRNPC, RBM39, LUC7L2 were specifically enriched in the E-Tn RNP compared to the E-dTn RNP (Table 1). These data suggested that the pyrimidine-rich sequence associates with pre-mRNA splicing factors, it may regulate the ratio of +KTS/−KTS isoforms by recruiting some of these splicing factors.

Figure 2.

Figure 2.

Open in a new tab

Affinity purification of RNPs assembled on in vitro transcribed RNA containing or lacking the pyrimidine-rich sequence. (A). Schematic of Tn-E and dTn-E constructs. Part of WT1 exon 9 and intron 9 containing or lacking the pyrimidine-rich sequence is cloned into a vector with T7 promoter. pA: polyadenylation signal. (B). Chromatography of in vitro assembled RNPs after fractionation. (C). Separation of purified RNPs on polyacrylamide gel. For Tn-E and dTn-E, in vitro transcribed RNAs are labeled with biotin and 32P. For Tn-c and dTn-c, RNAs were only labeled with 32P, which served as negative controls.

Table 1.

A subset of proteins present in Tn-E and dTn-E mRNPs

  Tn-E
 dTn-E
  
Gene Symbol Unique Total Unique Total Mol weight (kDa)
ACACA 75 221 135 261 266
HNRNPL 24 89 32 98 64
SKIV2L2 22 45 37 59 118
STRBP 19 74 41 78 74
ADAR 17 52 36 55 136
SRRT 13 44 27 42 101
HNRNPA1 8 39 10 39 39
SF3B1 6 15 11 12 146
SRSF3 4 13 5 7 19
SRSF6 5 15 7 8 40
SFRS9 0 0 4 4 26
TRA2A 5 17 4 4 33
TRA2B 0 0 7 8 34
ELAVL1 10 29 6 7 36
HNRNPC 14 64 3 4 34
RBM39 7 20 8 9 59
LUC7L2 5 15 4 4 47
Open in a new tab

HuR interacts with the pyrimidine-rich sequence and promotes the generation of −KTS isoform at the reporter level

To define which splicing factor could regulate the ratio of +KTS/−KTS isoforms, co-transfection of WT1-E9/10 reporter gene and plasmids expressing the splicing factors identified into HeLa cells were performed. Transfections with WT1 E9/10 reporter alone or together with the empty vector were used as controls. As showed in Figure 3A, co-transfection with empty vector led to 32% of −KTS isoform, over-expression of SRSF3, SRSF6, SRSF9, TRA2A, TRA2B, hnRNPC, RBM39, LUC7L2 and TIA1 resulted in no or only minor change of −KTS isoform (varied from 29% to 38%). In contrast, overexpression of HuR promoted the generation of −KTS isoform significantly (from 32% to 52%) (Fig. 3A). Western blot analysis was carried out to confirm the over-expression levels of indicated RNP components using HA antibody (Fig. 3B). Previously, it has been reported that HuR binds to U-rich element.33 To further investigate whether HuR interacts with pyrimidine-rich sequence in WT1 intron 9, we performed RNA immuno-precipitation (RIP) experiment using in vitro transcribed pre-mRNAs followed by RT-PCR. RNA containing the pyrimidine-rich sequence has much stronger association with HuR in comparison to control that lacks pyrimidine-rich sequence (Figure 3C). Taken together, these results supported that HuR bound to the pyrimidine-rich sequence and antagonized its effect in promoting the production of −KTS isoform at the reporter level.

Figure 3.

Figure 3.

Open in a new tab

HuR interacts with the pyrimidine-rich element and overexpression of HuR promotes the production of -KTS isoform at reporter level. (A). Proportion of -KTS isoform after co-transfection of indicated splicing factor with WT1 reporter. Percentage and SD are calculated on 3 independent experiments. (B). Western blot to show the protein level of over-expressed splicing factors. All the splicing factors contain HA tag, tubulin is used as loading control. NC: no co-transfection; Emp Vec: empty vector. (C). RNA immuno-precipitation (RIP) experiment showing HuR binds much stronger to RNA containing the pyrimidine-rich element. Lane 1and2: RNA input in RIP experiment; lane 3and4: RT-PCR products amplified from RNAs after RIP.

Overexpression of HuR increases the endogenous expression of −KTS isoform

To investigate whether HuR could influence the endogenous expression of +/−KTS isoforms, we performed transient transfection of HuR into 293T cells. Western blot analysis showed that HuR expression was enhanced at 24 hours after transfection (Fig. 4A), analysis of endogenous +/−KTS using RT-PCR revealed a mild increase on the expression of −KTS isoform (from 23% to 31%, Fig. 4B). To further verify the role of HuR in the production of endogenous −KTS isoform, we established several cell lines including 293T, TJ905 and T47D that stably expressed HA-tagged HuR. Western blot using HA or HuR antibody both confirmed the expression of HuR (Fig. 4C) in the stable cell line. Consistent with the results from transient transfection, a moderate increase of −KTS isoform was observed in 293T cell (from 18% to 28%), TJ905 cell (from 23% to 32%) and T47D cell (from 22% to 32%). These data indicated that transient or stable over-expression of HuR favored the endogenous expression of −KTS isoform.

Figure 4.

Figure 4.

Open in a new tab

Transient or stable overexpression of HuR enhances the proportion of endogenous -KTS isoform. (A). RT-PCR results showing transient over-expression of HuR promotes the production of endogenous -KTS isoform in 293T cells. Lower panel: western blot showing the level of HuR. Emp Vec: empty vector. WB: western blot. (B). Quantitation of -KTS isoform, The added up value of +KTS and -KTS forms was arbitrarily set to 100%. Data were showed as mean ± SD (n = 3). The p-values are calculated using T-test. SD: standard deviation. (C). RT-PCR results showing stable overexpression of HuR promotes the production of endogenous -KTS isoform in 293T, TJ905 and T47D cells. Lower panel: western blot showing the level of HuR. (D). Quantitation of -KTS isoform in stable cell lines. (n = 3). **: p ≤ 0.01 and ***: p ≤ 0.001.

HuR knockdown decreases the endogenous expression of −KTS isoform

To see whether HuR depletion could regulate the endogenous expression of −KTS isoform, we transfected HuR siRNA into 293T cells. As showed by western blot in Figure 5, HuR was efficiently depleted after siRNA treatment. A moderate decrease of endogenous −KTS isoform was observed (from 24% to 18%). Knockdown of HuR resulted in a more significant decrease of endogenous −KTS isoform in other cell lines including SW620 cell (from 21% to 11%), SNU-387 cell (from 31% to 18%) and AGS cell (from 44% to 30%). The results indicated that HuR played a role in the expression of −KTS isoform.

Figure 5.

Figure 5.

Open in a new tab

HuR depletion in 293T, SW620, SNU-387 or AGS cells results in decrease of -KTS isoform. (A). RT-PCR results showing knockdown of HuR decreases the production of endogenous -KTS isoform in 4 different cell lines. Lower panel: western blot showing the level of HuR after siRNA treatment. (B). Quantitation of -KTS isoform in different cell lines after HuR depletion. (n = 3). **: p ≤ 0.01 and ***: p ≤ 0.001.

Taken together, we conclude that HuR binds to the pyrimidine-rich cis-element in intron 9 and antagonizes its effect in regulating WT1 +/−KTS isoforms.

Discussion

Three amino-acids difference in WT1 +KTS/−KTS isoforms results in striking functional difference. The generation of +KTS/−KTS isoforms is due to the alternative usage of 2 adjacent 5’ splice sites at intron 9. However, mechanism on how the ratio of these 2 isoforms is regulated is poorly understood. This study shows that HuR is a trans-factor that regulates the production of +KTS/−KTS isoforms. It belongs to the ELAV/Hu family of RNA binding protein, it is ubiquitously expressed and essential for normal embryonic development.34,35

Previous study using PAR-CLIP identified about 26,000 binding sites of HuR in transcriptome, most of which were located in 3’ untranslated regions (UTR) or introns, implicating HuR may have important roles in splicing/mRNA processing.36 HuR preferentially binds to poly-U elements and AU-rich elements (AREs) in 3’ UTR and controls the stability of target mRNAs.37 Binding of HuR on introns regulates splicing of several genes including DST, ZNF207, PTBP2 and GANAB.38 Recently, several studies showed that HuR regulated alternative splicing by interacting with exonic splicing elements. HuR promoted FAS exon 6 skipping by interacting with an exonic splicing silencer,39 HuR also enhanced the inclusion of TRA2B exon2a by binding to the exon under oxidative stress.40 In addition, HuR was involved in the skipping of exon 11 of EIF4ENIF1 transcripts.41 These results together with our data indicate that in addition to its role in mRNA stability,42,43 HuR functions in alternative splicing.

Consistent with a previous study,44 deletion or partial replacement of the pyrimidine-rich sequence in intron 9 resulted in almost full shift to −KTS isoform, which suggested that this sequence was critical for the production of +KTS isoform. Furthermore, we identified that Tn sequence was widely conserved across vertebrates indicating it plays an important role (Fig. S1). As expected, further RNP purification followed by mass spectrometry revealed many splicing factors bound to this sequence. Among the splicing factors tested in this study, we did not identify any that favors the production of +KTS isoform. Even though many splicing related factors (eg. SRSF3, SRSF6, TRA2A) were specifically enriched in the E-Tn RNP compared to the E-dTn RNP, however, they didn’t affect +/−KTS splicing. One possible explanation is that the binding to the Tn sequence may be nonspecific. So it is critical to perform functional experiments to verify the results. Another explanation is that these splicing factors did bind to the Tn sequence, but didn’t regulate the splicing of +/− KTS. They may play various roles in other biologic processes. In contrast, HuR turned out to be a trans-factor that promoted the production of −KTS isoform. In addition, we demonstrated that HuR bound to the pyrimidine-rich sequence in WT1 intron 9. Overexpression of HuR resulted in an increase of −KTS isoform at reporter level as well as endogenous expression. Moreover, knockdown of HuR decreased the proportion of endogenous −KTS isoform. Thus, HuR binds to the pyrimidine-rich sequence and antagonizes its effect in regulating the ratio of +/−KTS isoforms.

However, compared to the striking effect at the deletion of the pyrimidine-rich sequence, the effect of HuR seems to be moderate, with ∼20% change observed at reporter level and ∼10% change in endogenous level. It is likely that additional cis-elements or trans-factors may also function in regulating the ratio of +/−KTS isoforms. In addition, although strong expression of HuR was detected after transient or stable over-expression, what actually counts in the regulation of alternative splicing might be the amount of protein with proper modifications or that assembled in the spliceosome rather than the overall expression level.

Finally, it will be interesting to investigate whether deletion of the pyrimidine-rich sequence from its natural genomic context could have similar effect on +/−KTS isoforms as observed in the reporter assay. With recent development of CRISPR/Cas9 technique,45,46 it is now possible to address the question.

Materials and Methods

Constructs and antibodies

WT1-E9/10 reporter was constructed by amplifying exon 9 and part of intron 9 of WT1 gene from HeLa genomic DNA using E9-F/E9-R primers listed in Suppl Table 1 and cloned into pCR3.1 vector at HindIII/XbaI sites followed by amplifying part of intron 9 and full exon 10 using E10-F/E10-R primers and cloned into XbaI/ApaI sites. The deletion mutants or mutants with sequence replacement were created by 2-step PCR as previously described.47 For Tn-E and dTn-E constructs, fragments were amplified from wild type E9/E10 reporter or the reporter lacking the pyrimidine-rich sequence using Bio-F/Bio-R primers and cloned into pcDNA3 vector at HindIII site. The primer sequences for cloning were listed in Suppl Table 1. All the constructs were verified by DNA sequencing. Splicing factors SRSF2, SRSF3, SRSF9, TRA2A, TRA2B, HuR, hnRNPC, RBM39, LUC7L2 and TIA1 were amplified from HeLa or 293T cDNA and cloned into modified pCI-neo vector (Promega, USA) containing HA tag at the 5’ end. Polyclonal antibody against HuR (Proteintech, 1:4000), monoclonal antibodies against HA tag (Covance, 1:2000), EGFP (Origene, 1:1000) and tubulin (Sigma, 1:5000) were used in western blots.

RT-PCR and detection of +/−KTS isoforms

For reverse transcription, 1ug total RNA was used for cDNA generation in a 25ul reaction with 200u M-MLV reverse transcriptase (Promega). To detect +/−KTS isoforms, cDNA was amplified with WT1 cDNA-F/cDNA-R primers listed in Suppl Table 2. The expected size of the PCR products is 101 bp for +KTS isoform and 92 bp for −KTS isoform. PCR products were first run on agarose gel and then separated on 6% nondenaturing polyacrylamide gel. Quantitation of band density was performed using ImageJ software.

Cell culture and transient transfection

HeLa cells were a gift from Reed Lab in Harvard Medical School and were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO2. Transient transfection was performed in 12-well plate with 1ug DNA of each plasmid using Lipofectamine 2000. Transfection efficiencies were monitored using EGFP as a control. For RNA interference, cells were plated in 12-well plate and 2ul siRNA at 50uM were transfected using Lipofectamine 3000 reagent. HuR siRNA was previously reported,48 non-target siRNA was used as negative control.

Purification of RNPs

RNPs were purified as previously described.49 Briefly, templates for in vitro transcription were amplified from Tn-E and dTn-E using primers IVT-F/IVT-R listed in Suppl Table 1. 1ug of the PCR product was transcribed with T7 RNA polymerase and labeled with 32P and biotin. RNPs were assembled by incubation of 2ug RNA with 600ul HeLa nuclear extract for 2h at 30°C in buffer containing 20 mM HEPES at pH 7.9, 100 mM KCl, 3.2 mM MgCl2, 20 mM creatine phosphate and 0.5 mM ATP. The mixture was fractionated using Sephacryl S-500 column, RNP fractions were pooled and followed by streptavidin purification. Eluted proteins were then precipitated using TCA and analyzed with mass spectrometry.

In vitro transcription and RNA immunoprecipitation

WT1-E9/10-pCR3.1 and WT1-dTn-pCR3.1 reporter minigenes were cut with ApaI, and RNAs were synthesized using T7 RNA polymerase (MEGAscript® Kit, Life) with 1ug linearized plasmid templates. Nuclear extract from 293T-HuR stable cell line were prepared as described.50 Following incubation of 25ng pre-mRNA with nuclear extract under splicing condition for 10 min at 30 °C, the reaction mixture were subjected to immunoprecipitation with Protein G agarose beads (GE Healthcare) coupled with anti-HA monoclonal antibody and 100ul of binding buffer (20mM HEPES at pH7.9, 500mM KCl, 0.1% Triton, 0.25 mM EDTA ). After rotation for 2h at 4°C, the immunoprecipitates were washed with 1ml binding buffer 6 times. Proteins were digested with Proteinase K (Merck, 10mg/ml) at 37°C for 10 min. RNAs were recovered by phenol/chloroform extraction and ethanol precipitation. RNAs were then reverse transcribed and analyzed by PCR using primers E9-F and E10-R listed in Suppl Table 2.

Stable cell line

HuR with HA tag was subcloned into pLVX-EF1a-IRES-Puro vector at EcoRI/XbaI sites and transfected into 293T cells together with pMD2G and pSPAKX vectors at a ratio of 8:1:1. Viral particles were harvested from the media 48h after transfection and infection to 293T, TJ905 and T47D cells was carried out followed by puromysin selection, colonies were expanded and verified for the expression of HuR.

Supplementary Material

2015RNABIOL0136R1-file004.pdf
2015RNABIOL0136R1-file003.pdf
2015RNABIOL0136R1-f06-z-4c.pdf

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by National Natural Science Foundation of China (Grant 81472491), Liaoning Pandeng Scholar Program and Innovative Research Team in University, Ministry of Education, China (No.IRT13049).

References

  • 1.Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF, Schroth GP, Burge CB. Alternative isoform regulation in human tissue transcriptomes. Nature 2008; 456:470-6; PMID:18978772; http://dx.doi.org/ 10.1038/nature07509 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Nilsen TW, Graveley BR. Expansion of the eukaryotic proteome by alternative splicing. Nature 2010; 463:457-63; PMID:20110989; http://dx.doi.org/ 10.1038/nature08909 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Haber DA, Buckler AJ, Glaser T, Call KM, Pelletier J, Sohn RL, Douglass EC, Housman DE. An internal deletion within an 11p13 zinc finger gene contributes to the development of Wilms' tumor. Cell 1990; 61:1257-69; PMID:2163761; [DOI] [PubMed] [Google Scholar]
  • 4.Haber DA, Sohn RL, Buckler AJ, Pelletier J, Call KM, Housman DE. Alternative splicing and genomic structure of the Wilms tumor gene WT1. Proc Natl Acad Sci U S A 1991; 88:9618-22; PMID:1658787; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Discenza MT, Vaz D, Hassell JA, Pelletier J. Activation of the WT1 tumor suppressor gene promoter by Pea3. FEBS Lett 2004; 560:183-91; PMID:14988020; http://dx.doi.org/8618823 10.1016/S0014-5793(04)00104-8; [DOI] [PubMed] [Google Scholar]
  • 6.Akpa MM, Iglesias DM, Chu LL, Cybulsky M, Bravi C, Goodyer PR. Wilms Tumor Suppressor, WT1, Suppresses Epigenetic Silencing of the beta-Catenin Gene. J Biol Chem 2015; 290:2279-88; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Montano G, Cesaro E, Fattore L, Vidovic K, Palladino C, Crescitelli R, Izzo P, Turco MC, Costanzo P. Role of WT1-ZNF224 interaction in the expression of apoptosis-regulating genes. Hum Mol Genet 2013; 22:1771-82; [DOI] [PubMed] [Google Scholar]
  • 8.Yang L, Han Y, Saiz FS, Minden MD. A tumor suppressor and oncogene: the WT1 story. Leukemia 2007; 21:868-76; [DOI] [PubMed] [Google Scholar]
  • 9.Baird PN, Groves N, Haber DA, Housman DE, Cowell JK. Identification of mutations in the WT1 gene in tumours from patients with the WAGR syndrome. Oncogene 1992; 7:2141-9; PMID:1331933; [PubMed] [Google Scholar]
  • 10.Cowell JK, Wadey RB, Haber DA, Call KM, Housman DE, Pritchard J. Structural rearrangements of the WT1 gene in Wilms' tumour cells. Oncogene 1991; 6:595-9; PMID:1851548; [PubMed] [Google Scholar]
  • 11.Bruening W, Bardeesy N, Silverman BL, Cohn RA, Machin GA, Aronson AJ, Housman D, Pelletier J. Germline intronic and exonic mutations in the Wilms' tumour gene (WT1) affecting urogenital development. Nat Genet 1992; 1:144-8; PMID:1302008; http://dx.doi.org/8618823 10.1038/ng0592-144; [DOI] [PubMed] [Google Scholar]
  • 12.Englert C, Vidal M, Maheswaran S, Ge Y, Ezzell RM, Isselbacher KJ, Haber DA. Truncated WT1 mutants alter the subnuclear localization of the wild-type protein. Proc Natl Acad Sci U S A 1995; 92:11960-4; PMID:8618823; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Rivera MN, Haber DA. Wilms' tumour: Connecting tumorigenesis and organ development in the kidney. Nat Rev Cancer 2005; 5:699-712; [DOI] [PubMed] [Google Scholar]
  • 14.Hosen N, Shirakata T, Nishida S, Yanagihara M, Tsuboi A, Kawakami M, Oji Y, Oka Y, Okabe M, Tan Bet al. The Wilms' tumor gene WT1-GFP knock-in mouse reveals the dynamic regulation of WT1 expression in normal and leukemic hematopoiesis. Leukemia 2007; 21:1783-91; PMID:17525726; http://dx.doi.org/10999598 10.1038/sj.leu.2404752; [DOI] [PubMed] [Google Scholar]
  • 15.Little M, Wells C. A clinical overview of WT1 gene mutations. Hum Mutat 1997; 9:209-25; PMID:9090524; http://dx.doi.org/10999598 10.1002/(SICI)1098-1004(1997)9:3<209::AID-HUMU2>3.0.CO;2-2; [DOI] [PubMed] [Google Scholar]
  • 16.Huff V. Wilms' tumours: about tumour suppressor genes, an oncogene and a chameleon gene. Nat Rev Cancer 2011; 11:111-21; PMID:21248786; http://dx.doi.org/10999598 10.1038/nrc3002; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Armstrong JF, Pritchard-Jones K, Bickmore WA, Hastie ND, Bard JB. The expression of the Wilms' tumour gene, WT1, in the developing mammalian embryo. Mech Dev 1993; 40:85-97;PMID:8382938; [DOI] [PubMed] [Google Scholar]
  • 18.Larsson SH, Charlieu JP, Miyagawa K, Engelkamp D, Rassoulzadegan M, Ross A, Cuzin F, van Heyningen V, Hastie ND. Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing. Cell 1995; 81:391-401;PMID:7736591 [DOI] [PubMed] [Google Scholar]
  • 19.Englert C, Vidal M, Maheswaran S, Ge Y, Ezzell RM, Isselbacher KJ, Haber DA. Truncated WT1 mutants alter the subnuclear localization of the wild-type protein. Proc Natl Acad Sci U S A 1995; 92:11960-4; PMID:8618823; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Rupprecht HD, Drummond IA, Madden SL, Rauscher FJR, Sukhatme VP. The Wilms' tumor suppressor gene WT1 is negatively autoregulated. J Biol Chem 1994; 269:6198-206; [PubMed] [Google Scholar]
  • 21.Ryan G, Steele- Perkins V, Mor ris JF, Rauscher FJR, Dressler GR. Repression of Pax-2 by WT1 during normal kidney development . Development (Cambridge, England) 1995; 121:867-75; [DOI] [PubMed] [Google Scholar]
  • 22.Wang ZY, Madden SL, Deuel TF, Rauscher FJR. The Wilms' tumor gene product, WT1, represses transcription of the platelet-derived growth factor A-chain gene. J Biol Chem 1992; 267:21999-2002; [PubMed] [Google Scholar]
  • 23.Drummond IA, Madden SL, Rohwer-Nutter P, Bell GI, Sukhatme VP, Rauscher FJR. Repression of the insulin-like growth factor II gene by the Wilms tumor suppressor WT1. Science (New York, N.Y.) 1992; 257:674-8; [DOI] [PubMed] [Google Scholar]
  • 24.Davies RC, Calvio C, Bratt E, Larsson SH, Lamond AI, Hastie ND. WT1 interacts with the splicing factor U2AF65 in an isoform-dependent manner and can be incorporated into spliceosomes. Gen Dev 1998; 12:3217-25; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kennedy D, Ramsdale T, Mattick J, Little M. An RNA recognition motif in Wilms' tumour protein (WT1) revealed by structural modelling. Nat Genet 1996;12:329-31; [DOI] [PubMed] [Google Scholar]
  • 26.Burwell EA, McCarty GP, Simpson LA, Thompson KA, Loeb DM. Isoforms of Wilms' tumor suppressor gene (WT1) have distinct effects on mammary epithelial cells. Oncogene 2007; 26:3423-30; PMID:17160023; http://dx.doi.org/10999598 10.1038/sj.onc.1210127; [DOI] [PubMed] [Google Scholar]
  • 27.Wang Z, Burge CB. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA 2008; 14:802-13; PMID:18369186; http://dx.doi.org/10999598 10.1261/rna.876308; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Chen M, Manley JL. Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat Rev Mol Cell Biol 2009; 10:741-54; PMID:19773805; http://dx.doi.org/10.1038 /nrm2777; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Chasin LA. Searching for splicing motifs. Adv Exp Med Biol 2007; 623:85-106; PMID:18380342; [DOI] [PubMed] [Google Scholar]
  • 30. Black DL. Mechanisms of alternative pre-messenger RNA splicing. Annual review of biochemistry. 2003;72:291-336; [DOI] [PubMed] [Google Scholar]
  • 31.Graveley BR. Sorting out the complexity of SR protein functions. RNA 2000; 6:1197-211; PMID:10999598; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Matlin AJ, Clark F, Smith CW. Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol 2005; 6:386-98; PMID:15956978; http://dx.doi.org/ 10.1038/nrm1645 [DOI] [PubMed] [Google Scholar]
  • 33.Busch A, Hertel KJ. Evolution of SR protein and hnRNP splicing regulatory factors. Wiley interdisciplinary reviews RNA. 2012; 3:1-12; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Zhu H, Hasman RA, Barron VA, Luo G, Lou H. A nuclear function of Hu proteins as neuron-specific alternative RNA processing regulators. Molecular biology of the cell. 2006;17:5105-14; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Katsanou V, Milatos S, Yiakouvaki A, Sgantzis N, Kotsoni A, Alexiou M, Harokopos V, Aidinis V, Hemberger M, Kontoyiannis DL. The RNA-binding protein Elavl1/HuR is essential for placental branching morphogenesis and embryonic development. Mol Cell Biol 2009; 29:2762-76; PMID:19307312; http://dx.doi.org/ 10.1128/MCB.01393-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Papadaki O, Milatos S, Grammenoudi S, Mukherjee N, Keene JD, Kontoyiannis DL. Control of thymic T cell maturation, deletion and egress by the RNA-binding protein HuR. J Immunol 2009; 182:6779-88; PMID:19454673; http://dx.doi.org/ 10.4049/jimmunol.0900377 [DOI] [PubMed] [Google Scholar]
  • 37.Lebedeva S, Jens M, Theil K, Schwanhausser B, Selbach M, Landthaler M, Rajewsky N. Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR. Mol Cell 2011; 43:340-52; PMID:21723171; http://dx.doi.org/ 10.1016/j.molcel.2011.06.008 [DOI] [PubMed] [Google Scholar]
  • 38.Mukherjee N, Corcoran DL, Nusbaum JD, Reid DW, Georgiev S, Hafner M, Ascano MJ, Tuschl T, Ohler U, Keene JD. Integrative regulatory mapping indicates that the RNA-binding protein HuR couples pre-mRNA processing and mRNA stability. Mol Cell 2011; 43:327-39; PMID:21723170; http://dx.doi.org/ 10.1016/j.molcel.2011.06.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Gonzales-van HS, Farrar JD. Interferon at the crossroads of allergy and viral infections. J Leukoc Biol 2015; 98(2):185-94; PMID:26026068; http://dx.doi.org/ 10.1189/jlb.3RU0315-099R [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Izquierdo JM. Hu antigen R (HuR) functions as an alternative pre-mRNA splicing regulator of Fas apoptosis-promoting receptor on exon definition. J Biol Chem 2008; 283:19077-84; PMID:18463097; http://dx.doi.org/ 10.1074/jbc.M800017200 [DOI] [PubMed] [Google Scholar]
  • 41.>Akaike Y, Masuda K, Kuwano Y, Nishida K, Kajita K, Kurokawa K, Satake Y, Shoda K, Imoto I, Rokutan K. HuR regulates alternative splicing of the TRA2beta gene in human colon cancer cells under oxidative stress. Mol Cell Biol 2014; 34:2857-73; PMID:24865968; http://dx.doi.org/ 10.1128/MCB.00333-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Chang SH, Elemento O, Zhang J, Zhuang ZW, Simons M, Hla T. ELAVL1 regulates alternative splicing of eIF4E transporter to promote postnatal angiogenesis. Proc Natl Acad Sci U S A 2014; 111:18309-14; PMID:25422430; http://dx.doi.org/ 10.1073/pnas.1412172111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Brennan CM, Steitz JA. HuR and mRNA stability. Cell Mol Life Sci 2001; 58:266-77; PMID:11289308; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Hinman MN, Lou H. Diverse molecular functions of Hu proteins. Cell Mol Life Sci 2008; 65:3168-81; PMID:18581050; http://dx.doi.org/ 10.1007/s00018-008-8252-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Yang C, Romaniuk PJ. The ratio of +/−KTS splice variants of the Wilms' tumour suppressor protein WT1 mRNA is determined by an intronic enhancer. Biochem Cell Biol 2008; 86:312-21; PMID:18756326; http://dx.doi.org/ 10.1139/o08-075 [DOI] [PubMed] [Google Scholar]
  • 46.Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337:816-21; PMID:22745249; http://dx.doi.org/ 10.1126/science.1225829 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013; 8:2281-308; PMID:24157548; http://dx.doi.org/ 10.1038/nprot.2013.143 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Lei H, Dias AP, Reed R. Export and stability of naturally intronless mRNAs require specific coding region sequences and the TREX mRNA export complex. Proc Natl Acad Sci U S A 2011; 108:17985-90; PMID:22010220; http://dx.doi.org/ 10.1073/pnas.1113076108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Pullmann RJ, Juhaszova M, Lopez DSI, Kawai T, Mazan-Mamczarz K, Halushka MK, Gorospe M. Enhanced proliferation of cultured human vascular smooth muscle cells linked to increased function of RNA-binding protein HuR. J Biol Chem 2005; 280:22819-26; PMID:15824116; http://dx.doi.org/ 10.1074/jbc.M501106200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Lei H, Zhai B, Yin S, Gygi S, Reed R. Evidence that a consensus element found in naturally intronless mRNAs promotes mRNA export. Nucleic Acids Res 2013; 41:2517-25; PMID:23275560; http://dx.doi.org/ 10.1093/nar/gks1314 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51. Folco EG, Lei H, Hsu JL, Reed R. Small-scale nuclear extracts for functional assays of gene-expression machineries. J Vis Exp 2012; PMID:22782264; doi: 10.3791/4140. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

2015RNABIOL0136R1-file004.pdf
krnb-12-12-1102831-s001.pdf (54.3KB, pdf)
2015RNABIOL0136R1-file003.pdf
krnb-12-12-1102831-s002.pdf (66.9KB, pdf)
2015RNABIOL0136R1-f06-z-4c.pdf
krnb-12-12-1102831-s003.pdf (304KB, pdf)

Articles from RNA Biology are provided here courtesy of Taylor & Francis

RESOURCES