Validation of ESRP-regulated exons reveals numerous large switches in exon splicing

To independently validate a large set of splicing changes predicted by the HJAY analysis, we predominantly used high-throughput capillary reverse transcription PCR (HT-RT–PCR) to validate nearly all simple cassette exons with constitutive flanking exons identified by the HJAYarrays (Venables et al, 2009). In the ESRP1 ectopic expression system, 158 of the 173 exons with successful HT-RT–PCR reactions demonstrated the HJAY predicted changes in exon inclusion of which 134 showed a higher confidence change of >5% in percent exon inclusion (Supplementary Table S3). In PNT2 knockdowns, 115 out of 183 successful HT-RT–PCR reactions yielded a lower overall validation rate, of which 71 exons had a >5% change in exon inclusion (Supplementary Table S4). We also used the HT-RT–PCR format to test 20 alternative 3′ or 5′ splice sites that were identified in the knockdown experiments and 11 validated the predicted change with a high confidence of >5% change between competing 5′ or 3′ splice sites (Supplementary Table S4). We also validated an additional 13 HJAY predicted splicing changes using traditional RT–PCR, including cases that involved predicted mutually exclusive exons (Supplementary Figure S1). In total, we validated 210 predicted changes in splicing with a >5% change in splice isoforms.

Exons showing the overall largest validated changes in exon inclusion identified in each experimental approach are shown in Table I. Among the validated events, were 22 ‘switch-like' changes in exon splicing in which knockdown or ectopic expression induced a switch from predominant exon inclusion (>66%) to mostly exon skipping (<34% inclusion), or vice versa as defined in Xing and Lee (2005). Also noteworthy were 19 examples in which one splice variant was predominant at baseline in each cell type, but where knockdown or ectopic ESRP expression induced expression of a second splice variant. Even though such changes were less complete, they nonetheless predicted expression of numerous epithelial- and mesenchymal-specific variants (e.g. ARFGAP2, ARHGEF11). RALGPS2 and ITGA6 demonstrated large changes in ESRP-mediated exon inclusion in both experimental systems (Figure 2A and B). In MAGI1, ESRP expression promoted exon skipping in both the ESRP overexpression and knockdown analysis (Figure 2C). In addition, we noted that two different exons in MAP3K7 (also known as TAK1) transcripts were either enhanced or silenced by the ESRPs (Figure 2D). Of note, regulated exons in ITGA6 and MAP3K7 are alternative penultimate exons that generate different C-terminal domains in the resulting protein isoforms. Similar to the ITGA6 exon, we identified splicing switches in a number of other penultimate exons containing stop codons (e.g. in MPRIP, CD46, STX2, and STX3 transcripts). As most of these stop codons are <50–55 nt upstream of the terminal intron, these transcripts are predicted to elude RNA degradation through the nonsense-mediated decay pathway and this type of exon switch provides an effective means of changing the C-terminal domain. Many of these splicing switches occur in transmembrane proteins, indicating that this might be a common mechanism of altering the intracellular signalling pathways of integral membrane receptors and matrix binding proteins. To further verify that changes in splicing were the result of ESRP knockdown, we also validated a subset of splicing switches using a second combination of siRNAs directed against different sequences in ESRP1 and ESRP2. We also performed a ‘rescue' using the RNAi resistant mouse Esrp1 cDNA. This analysis confirmed that these splicing switches are specifically due to ESRP1 and ESRP2 knockdown and not because of off-target effects (Figure 2E and F).

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Figure 2

Robust examples of validated ESRP-induced enhancement and silencing of microarray predicted target exons. (A) Validation of an ESRP-enhanced penultimate exon in ITGA6. The UCSC browser view of the transcripts that contain or skip the exon is shown at the top. In the centre are HJAY array data showing changes in probeset intensity in response to ectopic expression of Esrp1 (MDA-MB-231 cells) or knockdown of ESRP1 and 2 (PNT2). In each panel, the red line indicates changes in total transcript expression levels based on probesets in constitutive exons. The blue lines represent average changes detected by each of the eight probes in each probeset against each exon or exon–exon junction. UP denotes upstream junction; DOWN denotes downstream junction; SKIP denotes junction formed by exon skipping. Shown at the bottom are electropherograms from HT-RT–PCR validation assays with gel peaks corresponding to exon inclusion and skipping boxed. Exon inclusion levels are given as percentages for each condition. This format also applies to (B–D). (B) Validation of an ESRP-enhanced exon in RALGPS2. (C) Validation of an ESRP-silenced exon in MAGI1. (D) Validation of opposite changes in splicing of two exons in the MAP3K7 transcript in response to ESRP knockdown. (E) RT–PCR was used to assay splicing of select genes in PNT2 cells treated with two different sets of siRNAs (A, B) directed against both ESRP1 and ESRP2 or cells treated with siRNA-set A and expressing an siRNA-resistant cDNA directing expression of mouse Esrp1 protein. (F) Western blot demonstrating expression of the Flag-tagged mouse Esrp1 protein.

Table 1

Exons with the largest validated positive and negative change in splicing upon ESRP knockdown or ectopic expression

MDA-MB-231: ectopic expression of Esrp1
Gene symbol	Gene name	Exon coordinates	% Inclusion	% Inclusion	% Change
			EV control	Esrp1
ESRP-enhanced exons
VPS39	Vacuolar protein sorting 39	chr15:40271556–40271588	1.2	100.0	98.8
FNIP1	Folliculin-interacting protein 1	chr5:131074170–131074253	3.1	88.4	85.3
UAP1	UDP-N-acetylhexosamine pyrophosphorylase	chr1:160829149–160829196	4.4	86.5	82.2
KIF13A	Kinesin family member 13A	chr6:17879324–17879428	2.1	79.1	77.1
RALGPS2a	Ral GEF with PH domain and SH3-binding motif 2	chr1:177127988–177128065	6.4	80.9	74.5
ITGA6	Integrin α-6	chr2:173074746–173074875	30.0	96.4	66.4
KIAA0494	KIAA0494	chr1:46926612–46926803	25.8	91.5	65.7
ARHGEF12	Rho guanine nucleotide exchange factor 12	chr11:119785313–119785369	18.2	82.1	63.9
C10orf137	Chromosome 10, open reading frame 137	chr10:127407562–127407663	37.3	100.0	62.7
BTN2A1	Butyrophilin subfamily 2 member A1	chr6:26571004–26571104	17.9	76.6	58.7
ESRP-silenced exons
MAGI1a	Membrane-associated guanylate kinase, WW and PDZ 1	chr3:65408737–65408772	92.5	1.9	−90.6
PLAA	Phospholipase A-2-activating protein	chr9:26907095–26907163	97.0	7.9	−89.1
LAS1L	LAS1-like protein	chrX:64670215–64670340	100.0	16.1	−83.9
LRRFIP2	Leucine-rich repeat flightless-interacting protein 2	chr3:37107962–37108033	88.2	16.1	−72.1
ELOVL1	Elongation of very long chain fatty acids protein 1	chr1:43603444–43603634	80.9	9.4	−71.4
SPINT2	Serine peptidase inhibitor, Kunitz type, 2	chr19:43466107–43466277	87.6	18.9	−68.7
ANKRD26	Ankyrin repeat domain-containing protein 26	chr10:27390079–27390177	100.0	36.1	−63.9
SOD2	Superoxide dismutase 2	chr6:160029148–160029264	85.3	25.9	−59.4
PPFIBP1	PTPRF-interacting protein-binding protein 1	chr12:27721264–27721296	62.7	3.4	−59.3
HMBS	Hydroxymethylbilane synthase	chr11:118468324–118468443	93.3	34.6	−58.7
PNT2 cells: combined knockdown of ESRP1 and ESRP2b
Gene symbol	Gene name	Exon coordinates	% Inclusion	% Inclusion	% Change
			siGFP	siESRP1/2
ESRP-enhanced exons
SLC37A2	Solute carrier family 37 member 2	chr11:124461310–124461366	80.2	3.7	−76.5
RALGPS2a	Ral GEF with PH domain and SH3-binding motif 2	chr1:177127988–177128065	76.5	10.7	−65.8
ITGA6	Integrin α-6	chr2:173074746–173074875	87.8	27.5	−60.3
GOLGA2	Golgin subfamily A member 2	chr9:130069294–130069374	60.8	21.5	−39.3
MYO9A	Myosin-IXa	chr15:69972360–69972572	89.6	50.9	−38.7
TCF7L2	Transcription factor 7-like 2	chr10:114714305–114714373	44.7	15.5	−29.2
MAP3K7	Mitogen-activated protein kinase kinase kinase 7 (TAK1)	chr6:91310992–91311072	31.3	5.2	−26.1
MPZL1	Myelin protein zero-like protein 1	chr1:166011925–166012027	91.9	66.2	−25.7
HISPPD1	Histidine acid phosphatase domain-containing protein 1	chr5:102546834–102547007	30.0	6.8	−23.2
INTS9	Integrator complex subunit 9	chr8:28760183–28760245	95.7	73.5	−22.2
ESRP-silenced exons
COL16A1	Collagen α-1(XVI)	chr1:31917992–31918039	24.9	70.8	46.0
PLAA	Phospholipase A-2-activating protein	chr9:26907095–26907163	50.7	95.6	44.9
MAP3K7	Mitogen-activated protein kinase kinase kinase 7 (TAK1)	chr6:91284887–91285002	27.5	71.6	44.1
PQLC3	PQ-loop repeat-containing protein 3	chr2:11232545–11232586	49.2	91.0	41.8
JAG2	Jagged-2	chr14:104688373–104688486	33.0	72.4	39.4
MAGI1a	Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 1	chr3:65408737–65408772	7.0	44.9	37.9
PRC1	Protein regulator of cytokinesis 1	chr15:89313313–89313354	27.3	63.3	36.1
MEST	Mesoderm-specific transcript homolog	chr7:129927866–129927967	46.6	81.6	35.0
UBE2K	Ubiquitin-conjugating enzyme E2 K	chr4:39455697–39455825	57.6	92.4	34.8
TBC1D23	TBC1 domain family member 23	chr3:101513367–101513411	31.5	64.5	33.0
The 10 cassette exons with the largest validated increase or decrease in percent change in exon inclusion in each experimental approach as determined by HT-RT–PCR. Values are shown in response to expression of a murine cDNA for Esrp1 in mesenchymal MDA-MB-231 cells or upon combined knockdown of ESRP1 and ESRP2 in epithelial PNT2 cells.
aNote that RALGPS2 and MAGI1 were among the top 10 enhanced and silenced exons that were validated in both experimental approaches.
bIn PNT2 cells most values represent mean from two RT–PCRs.

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Identification of an ESRP-binding motif within ESRP-regulated exons and intronic flanking sequences

We previously demonstrated that ESRP1 binds the FGFR2 auxiliary cis-element ISE/ISS-3, located in the intron between mutually exclusive exons IIIb and IIIc, which implied that the ESRPs promoted splicing of the upstream exon IIIb while silencing the downstream exon IIIc (Warzecha et al, 2009a). To define a consensus ESRP-binding motif, we searched for conserved hexamers in the introns flanking validated ESRP-regulated cassette exons as well as in the exons themselves. The top five motifs in each position relative to enhanced or silenced exons are presented in Figure 3A (complete motif analysis is shown in Supplementary Table S5). We noted a remarkably enriched motif downstream of ESRP-enhanced exons consisting of hexamers that contained repeats of UGG or GGU; similar to the FGFR2-binding site and consistent with a model in which ESRP-binding downstream of an exon promotes its inclusion. Similar motifs were enriched within ESRP-silenced exons. As UGG was a core of the most enriched motifs and to account for some degeneracy in the binding site, we mapped the 5-mer motifs UGGUG, GGUGG, and GUGGU across the set of validated ESRP-regulated exons within the windows used to derive the motifs (Supplementary Figure S2A and B). We noted that clusters of these sequences were enriched downstream of enhanced exons. In contrast, we observed enrichment of these motifs within ESRP-silenced exons, whereas these motifs were largely absent in ESRP-enhanced exons. We also noted that these 5-mers were present in greater numbers upstream of silenced exon than the enhanced exons. Therefore, the trend of these data was consistent with a ‘map' in which ESRP-binding downstream of a regulated exon promotes exon inclusion and binding within the exon, and possibly the upstream intron, promotes exon skipping. We also note that although there are a number of ESRP-enhanced exons in which the motif was not identified within the 250-nt window used here, we often noted UGG-rich motifs in conserved sequence tracks located further downstream of these exons (e.g. ENAH). To determine whether the UGG-rich motifs are direct binding targets for the ESRPs, we performed RNA electrophoretic mobility shift assay (EMSAs) using recombinant ESRP1 protein. We tested whether the protein bound to RNA sequences containing repeats of UGG compared with UCG repeats. These experiments validated sequence-specific binding to the UGG repeat motifs, but not to those in which the motif is disrupted (Figure 3B). EMSA titration analysis to determine binding affinities revealed a dissociation constant (Kd) of 31.7 nM, indicating that this motif represents a bona fide high-affinity target for the ESRPs (Figure 3C).

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Figure 3

UGG motifs define a high-affinity ESRP-binding site with position-dependent functions. (A) Motif enrichment analysis in the exon and flanking 250 nt of intronic sequence, excluding the splice sites. The top five most enriched motifs ranked by Fisher P-value (by nucleotide) in each window for ESRP-enhanced and ESRP-silenced exons are shown. UGG-containing motifs are boxed. The Fox-binding site is indicated with an asterisk. (B) Esrp1 binds specifically to a UGG repeat-containing RNA. Increasing concentrations of recombinant GST-tagged Esrp1 were bound to UGG X 15 radiolabelled RNA or a mutated UCG X 15 control and resolved on a non-denaturing polyacrylamide gel. (C) Determination of Esrp1-binding affinity to the UGG repeats using increasing concentrations of Esrp1 (0–108 nM). The percent of bound RNA was plotted against the concentration of Esrp1 and fit to a sigmoidal curve to determine the dissociation constant (Kd).

We noted that the Fox-binding site UGCAUG was one of the top conserved hexamer motifs downstream of both ESRP-enhanced and ESRP-silenced exons. As the presence of this motif downstream of an exon predicts Fox-mediated enhanced exon inclusion, this observation suggests that the Fox proteins and the ESRPs can function both cooperatively and antagonistically in the regulation of exon splicing, although further studies are needed to investigate functional interactions between these regulators (Zhang et al, 2008). However, we did note a number of ESRP-regulated exons that were previously identified as Fox-regulated exons and these included examples in which these splicing regulators promoted the same as well as opposite effects on exon splicing (Supplementary Table S6).

The position of ESRP-binding motifs define an epithelial splicing regulatory ‘RNA map'

To validate that the ESRPs can directly regulate splicing by binding to UGG-rich motifs, we constructed minigenes corresponding to two alternative exons in the ITGA6 and RALGPS2 transcripts that are robustly enhanced by ESRP expression. These exons and conserved flanking intronic sequences were inserted between two adenoviral exons. Both gene transcripts contain a highly conserved sequence element downstream of the exon that contains several putative ESRP-binding sites (Figure 4A and B). We co-transfected these minigenes in 293T cells, which do not express ESRP, with a plasmid directing expression of ESRP1 or empty vector control. Exon inclusion was significantly increased in both minigenes when ESRP1 was co-expressed (Figure 4C). We introduced point mutations in both minigenes to disrupt conserved UGG motifs (Figure 4A and B). In ITGA6 mutations in the UGG motifs abolished the ability of ESRP1 to promote exon inclusion, whereas mutations outside of these motifs did not. In the case of RALGPS2 splicing of the exon containing the mutated sequence motifs was reduced even in the control empty vector co-transfections. This could be due either to reduced binding of other GU-rich binding proteins that may also promote exon inclusion, or through the creation of a silencing element. Nonetheless, these mutations similarly abrogated the ability of ESRP1 to promote exon inclusion (Figure 4C). We validated that these ITGA6 and RALGPS2 sequence elements bind ESRP1 directly by EMSA using RNA probes from the sequences presented in Figure 4A and B. Mutations in the ITGA6 element targeting UGG motifs nearly abolished ESRP1 binding, whereas the mutations in the RALGPS2 element disrupted binding, albeit to a lesser extent (Figure 4D).

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Figure 4

The ESRPs regulate splicing through direct binding to UGG-rich motifs. (A) ITGA6 exon 25 and flanking intronic sequences were inserted into the intron of a minigene reporter. A conserved UGG-rich element downstream of the exon is indicated by a grey box and point mutations both within (Mut1) and outside (Mut2) of UGG motifs are indicated. Numbers above this and subsequent minigenes indicate the number of nucleotides upstream of the 3′ splice site and downstream of the 5′ splice site. (B) Schematic of the RALGPS2 exon 15 minigene. (C) ESRP-mediated enhancement of exon inclusion requires the wild-type (WT) UGG-rich elements downstream of the ITGA6 and RALGPS2 exons, but does not occur when they are mutated (Mut1). Mutations outside these motifs (Mut2) do not abrogate ESRP-mediated exon splicing. RT–PCR analysis monitoring levels of exon inclusion (I) versus exclusion (E) in 293T cells co-transfected with the respective minigenes and an empty vector control (EV) or vector expressing Esrp1. Data are presented as the average exon inclusion percentage and s.d. from three independent transfections. At bottom are western blots validating expression of FLAG-tagged Esrp1 protein. (D) EMSA comparing binding of Esrp1 to the WT ITGA6 and RALGPS2 sequence elements and to the respective mutated sequences. Concentrations of Esrp1: 0, 54, 81, 108, 161, and 1075 nM. (E) Diagram of the OSBPL3 exon 9 minigene. Conserved UGG-rich elements occur in both the upstream intron (lower case) and within the exon (upper case letters). Point mutations were introduced in the intron (Int mut), exon (Ex mut), and both together. (F) ESRP-mediated exon silencing OSBPL3 exon 9 involves contributions by both upstream intronic and exonic UGG-rich cis-elements. RT–PCR analysis is as described in (C). (G) Diagram of the MAGI1 exon 7 minigene. Sequence alignment of the element in five vertebrate species is shown below the sequence element in which UGGs are boxed. (All UGG-rich motifs presented in this figure are highly conserved, but for space reasons the data are limited to this example). (H) ESRP-mediated exon silencing requires UGG-rich elements in the introns upstream of the MAGI1 exon. RT–PCR analysis is as described in (C).

We next investigated whether the ESRPs promote exon skipping through binding to UGG-containing motifs in the upstream introns or within the exon itself as predicted by the motif analysis. We constructed a minigene corresponding to an alternative exon in the OSBPL3 transcript that is silenced by the ESRPs. There are putative ESRP-binding sites both directly upstream of and within the alternative exon (Figure 4E). Point mutations were introduced to disrupt the UGG motifs in the upstream intron, in the exon, or in both regions. Exon inclusion was greatly reduced when ESRP1 was co-expressed with the wild-type minigene (Figure 4F). ESRP1 was able to silence exon inclusion when the exonic motifs were mutated, but to a lesser extent compared with wild type. Interestingly, mutation of the intronic motifs alone had a negligible effect on the ability of ESRP1 to promote exon silencing, but when both sets of mutations are made in the minigene, ESRP1-mediated silencing of this exon is nearly abolished. These results suggest that silencing of OSBPL3 exon 9 by the ESRPs is promoted by binding to both sites, although binding at the exonic sites has a predominant function. To further examine whether the ESRPs can silence splicing through binding to upstream intronic sites alone, we constructed a minigene corresponding to the alternative exon in MAGI1 that is strongly silenced by ESRP expression. Here, we noted a highly conserved UGG-containing cis-element upstream of the alternative exon, whereas no clear candidate motifs were present in the exon itself (Figure 4G). Co-transfections revealed that ESRP1 significantly reduced exon inclusion levels in the wild-type minigene but that the UGG mutations essentially abolished ESRP1-mediated silencing activity (Figure 4H). Collectively, these results provide evidence that the ESRPs directly regulate splicing of these exons and strongly implicate UGG-containing motifs as components of ESRP-binding sites that mediate position-dependent splicing regulation.

Sustained ESRP knockdown in mammary epithelial cells induces changes in epithelial–mesenchymal cell markers and morphology consistent with an EMT

To determine whether prolonged disruption of this broad ESRP-regulated splicing programme might induce changes in cell morphology or phenotype indicative of an EMT programme, we transduced human mammary epithelial cells (HMECs) with lentiviruses expressing shRNAs targeting ESRP1, ESRP2, or both proteins together. These shRNAs effectively reduced the levels of each protein (Figure 5A). As expected, prolonged knockdown of the ESRPs resulted in changes of exon inclusion in ESRP-target transcripts (Figure 5B). We noted little or no change in splicing upon knockdown of ESRP2 alone, whereas ESRP1 knockdown was associated with some changes in splicing and combined knockdown generally achieved the largest change, suggesting that ESRP1 is a more robust splicing regulator. To confirm that these changes in splicing were specific for the defined set of ESRP targets, we also tested several unrelated alternatively spliced exons that showed no change in exon inclusion levels (Supplementary Figure S3). Strikingly, HMECs in which ESRP1 alone or both ESRPs were depleted displayed altered cellular morphology, with a loss of typical epithelial cobblestone appearance and acquisition of an elongated spindle shape compared with control cells (Figure 5C). HMECs expressing the ESRP2 shRNA retained the morphology of the control cells (data not shown). To assess whether these morphological changes were indicative of an EMT, we analysed for changes in expression of several prototypical EMT markers. Immunoblotting showed that there was a corresponding induction of protein expression for the mesenchymal markers vimentin, fibronectin, N-cadherin, and α-SMA with ESRP1 or combined ESRP1 and ESRP2 knockdown (Figure 5D). We also observed increased matrix metalloprotein-2 activity upon ESRP1 or combined knockdown by in gel zymography; further evidence of invasive mesenchymal cell properties (Figure 5E) (Liu et al, 2009). However, there was negligible if any reduction in E-cadherin protein expression or that of other epithelial cell-specific markers (Figure 5D and data not shown). Nonetheless, immunofluorescence analysis of E-cadherin protein showed that ESRP knockdown induced a generalized loss of E-cadherin localization from the plasma membrane at sites of cell–cell junctions and relocalization in a diffuse cytoplasmic pattern, consistent with changes in E-cadherin during the EMT in other model systems (Figure 5F). To investigate the phenotypic consequences of ESRP depletion, we used the scratch wound assay, which demonstrated that a loss of the ESRP-splicing programme promoted a modest, but significant increase in cell motility, consistent with the acquisition of mesenchymal cell properties (Figure 5G and H). Together, these results demonstrate that disruption of the epithelial-splicing programme, regulated predominantly by ESRP1, is sufficient to induce epithelial cells to undergo some of the changes in gene expression and morphology that are associated with the EMT. We note that although ESRP loss is associated with an increase in expression of several mesenchymal markers, there was not an overt decrease in expression of epithelial markers such as E-cadherin. These findings thus indicate that even in the absence of a complete switch in the transcription of several known EMT markers, these global changes in splicing can induce some of the transitions in cell behaviour that are observed during the EMT. These studies also support our hypothesis that the ESRP-regulated splicing programme is an essential feature of the epithelial phenotype and that many changes in cell behaviour associated with the EMT are due to alterations in the functions of proteins that undergo isoform switching during this process.

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Figure 5

Identification of a discrete ESRP-regulated splicing signature that can distinguish epithelial from mesenchymal cell types. (A) Western blot using a monoclonal antibody that recognizes both ESRPs to validate specific shRNA-mediated depletion in the nucleus of HMEC cells (B) Switches in splicing of ESRP-target genes after prolonged knockdown of each ESRP alone or in tandem. RT–PCR was used to assay alternative splicing in the HMEC samples described in (A). Percent exon inclusion is indicated beneath each lane. (C) Prolonged knockdown of ESRP1 or ESRP1 and 2 induces morphological changes in HMEC cells. Phase-contrast images illustrate that cells depleted of ESRP1 and 2 or ESRP1 alone undergo EMT-like changes in morphology including a reduction in cobblestone appearance and cell–cell contacts and increased stellate-shaped cells. (D) Western blot demonstrating pronounced increase in expression of mesenchymal protein markers in cells depleted of ESRP1 or ESRP1 and 2 but not ESRP2 alone compared with control cells. (E) An increase in matrix metalloprotein-2 (MMP-2) activity accompanies the increase in expression of mesenchymal proteins. Conditioned media were collected and analysed by gelatin zymography to detect MMP-2 secretion. (F) Immunofluorescence staining of E-cadherin (red) shows pronounced localization near the plasma membrane in control cells and diffused cytoplasmic localization in cells depleted of ESRP1 or ESRP1 and 2. Nuclei are stained with DAPI. (G) Prolonged knockdown of ESRP1 alone or ESRP1 and ESRP2 results in increased cell motility. The scratch wound-healing assay was used to monitor migration of the HMEC samples shown in (C). (H) The scratch assay was repeated with nine replicates each for HMEC shRNA control and combined shRNAs against ESRP1 and ESRP2 and time to complete wound closure determined. Wound closure time was significantly shorter for cells depleted of both ESRPs compared with the control cells. Each bar represents the mean±s.d. of replicate samples (P-value determined by Welch two-sample t-test). (I) Heat map illustrating exon inclusion levels from select genes across a panel of epithelial and mesenchymal cell lines as measured by HT-RT–PCR. (J) Switches in splicing of the ‘EMT signature' exons during Twist-induced EMT in HMEC cells. RT–PCR was used to assay alternative splicing in HMEC cells transduced with empty vector (EV) or Twist expressing retroviruses. Percent exon inclusion is indicated beneath each lane.

HJAY microarray target preparation and hybridization

RNA purification, preparation of biotinylated sense-strand DNA, and hybridization was carried out as described for exon array analysis (Warzecha et al, 2009b). The CEL file data sets can be accessed at NCBI GEO repository through accession number GSE22937.

RT–PCR and HT-RT–PCR

Quantification of splicing by standard RT–PCR and high-throughput RT–PCR at the Université de Sherbrooke was performed as described (Warzecha et al, 2009b). For complex splicing events, restriction digestion and/or DNA sequencing was used to validate PCR product identity. Complete HT-RT–PCR data can be accessed at http://palace.lgfus.ca.

Antibodies and immunoblotting

Cell lysates were resolved on 4–12% NuPAGE Bis-tris gels (Invitrogen) and immunoblotted as described (Warzecha et al, 2009a). Antibodies used were anti-β actin (Sigma AC-15, 1:1000), anti-Flag (Stratagene, 1:1000), anti-Vimentin (Thermo Scientific Ab2 Clone V9, 1:150), anti-Fibronectin (BD Transduction, 1:10 000), anti-smooth muscle actin (Sigma, 1:300), anti-N-cadherin (BD Transduction, 1:250), and anti-E-cadherin (Cell Signaling Technology, 1:1000). ESRP1 antibodies that specifically detect ESRP1 (clone 27H12) and both ESRP1 and ESRP2 (clone 23A7) were obtained as custom monoclonal antibodies against our Glutathione-s-transferase (GST)-Esrp1 recombinant protein as prepared by Rockland Immunochemicals, Inc. (Gilbertsville, PA). These antibodies were shown to be specific with no detectable cross-reactivity against the closest RNA-binding protein homologs GRSF1, hnRNPF, or hnRNPH1.

Recombinant protein production and RNA EMSA analysis

Production of recombinant GST-Esrp1 fusion protein from plasmid GST-Esrp1-FL was performed using standard techniques and purified using Glutathione sepharose beads (GE healthcare). Sequences for in vitro transcription were inserted into the ClaI-Xho restriction sites in plasmid pDP19RC-ΔEE and transcription by T7 polymerase (Ambion) after XhoI digestion was carried out as described (Hovhannisyan and Carstens, 2007). [³²P]UTP-radiolabelled RNAs were used at a specific activity of 2.4 × 10⁸ CPM/μg and 50 000 CPM (~8–13 fmol) of radiolabelled RNA and GST-Esrp1 were incubated in a 10-μl binding reaction (16 mM Hepes pH 7.9, 95 mM KCl, 2 mM MgCl₂, 60 μM EDTA, 1 mM DTT, 6% glycerol, 0.1 mM heparin (Sigma #H-3393), and 0.15 mM PMSF). Binding reactions were incubated at 30°C for 30 min and loaded on 4% non-denaturing Tris-borate acrylamide gels and electrophoresed at 4°C for 1.5 h at 200 V. Quantification for Kd determination was performed using a PhosphorImager (Molecular Dynamics) and the % RNA bound was determined by calculating the ratio of [(shifted complexes)/(free RNA+shifted complexes)]. Data were analysed using the dose response with variable slope regression equation in Prism software and the Kd was determined using the EC50 value corresponding to the concentration of ESRP1 at 50% binding saturation.

In gel zymography

Cells were cultured in serum-free MEGM (Lonza) for 24 h. Cell culture media was lyophilized to dryness and rehydrated with SDS loading buffer (10% SDS, 50% glycerol, 0.4 M Tris, pH 6.8, and 0.1% bromophenol blue) and separated on 8% polyacrylamide/0.3% gelatin gels. Gels were then washed in 0.01% Triton X-100, 1 h rocking gently, and incubated in reaction buffer (50 mM Tris, pH 8.0, and 5 mM CaCl₂) at 37°C for 24 h. After the reaction, gels were stained with staining buffer (0.12% Coomassie blue R-250, 50% methanol, and 20% acetic acid) for 1 h and destained overnight with destaining buffer (22% methanol and 10% acetic acid). Gel loadings were normalized to total protein measured with a Bio-Rad Protein Assay (Richmond, CA).

Immunofluorescence

Transduced HMEC cells were grown on coverslips, washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde at room temperature for 12 min, washed, permeabilized for 5 min with PBS+0.2% Triton X-100, and blocked for 10 min with 10% goat serum in PBS. The coverslips were incubated sequentially with anti-E-cadherin primary antibody (1:150) and Texas Red-conjugated anti-Rabbit secondary antibody (1:4000) and counterstained with DAPI blue.

We thank Don Baldwin, Yi Li, and John Hogenesch for technical support and discussion; Kristen Lynch for review of the manuscript; and the University of Iowa ICTS for computer support. This research was supported by NIH grants R01-CA093769 and R01-GM088809 (RPC), R01-HG004634 and Edward Mallinckrodt, Jr Foundation (YX), R01-GM085146 (WG), and T32-GM08216 (CCW).