androgens and other steroid hormones are implicated in the post- transcriptional regulation of gene expression [9,10,11,12,13]. AR has also been shown to modulate expression of mRNA isoforms regulated by alternative pre-mRNA splicing and promoter selection [14,15]. In PCa, there is emerging evidence to suggest that expression of specific splice isoforms derived from cancer- relevant genes may underlie tumour biology and contribute to clinical disease progression [15]. Several genome-wide studies have successfully identified androgen-regulated transcriptional events using PCa models [16,17,18,19,20,21,22], but have been unable to discern between alternative mRNA species due microarray design or platform- specific analysis issues. Newer splicing-specific microarrays and high throughput sequencing (mRNA-Seq) approaches theoretical- ly allow identification of all mRNA isoforms as well as determination of gene expression in PCa [15]. An mRNA-Seq- based analysis of the androgen-responsive LNCaP cell transcrip- tome [22] was able to detect important differences in gene expression, but did not achieve the required tag density to identify quantitative changes in mRNA isoform abundance. In this study, our objective was to identify androgen-regulated alternative mRNA isoforms generated by alternative splicing and from alternative transcriptional initiation sites due to promoter selection. Screening for gene expression changes at exon-level resolution also enabled us to identify novel AR-regulated genes and associated pathways. In particular, we identified clusters of novel androgen-regulated genes associated with, and including the tyrosine kinase LYN, and a novel mRNA isoform derived from the Tuberous Sclerosis Complex TSC2 tumour suppressor gene under the control of an AR-dependent alternative internal promoter. This novel TSC2 mRNA contains the reading frame for only the carboxy (C)-terminal region of the tuberin protein, and was predicted to lack an interaction domain required for heterodimer- isation with hamartin to enable inhibition of the mammalian target of rapamycin (mTOR) pathway, which is commonly dysregulated in cancer. Results Exon-specific analyses of the androgen-responsive LNCaP transcriptome identifies novel androgen- regulated genes and mRNA isoforms To monitor the global response of the LNCaP transcriptome to androgen stimulation at single exon-level resolution, total RNA was extracted from LNCaP cells grown in steroid-deplete medium (n = 4) and following 24 hours of treatment (n = 4) with 10 nM synthetic androgen analogue R1881. The 24 hour time point is consistent with a recently published kinetic analysis of androgen- regulated gene expression, which reported a robust genome-wide transcriptional response to androgen stimulation after 24 hours [17]. To confirm an AR-dependent transcriptional response, we used qPCR to demonstrate up-regulation of KLK3 gene expression in each of the treatment samples as compared with controls (Figure S1). Using the Human Exon 1.0 ST Array, we profiled expression of more than one million exons in a full genome-wide screen to identify changes in the specific exon content of mRNA transcripts as well as overall gene expression. We identified a total of 287 genes that were up-regulated by at least 2-fold in response to androgen treatment and 263 genes that were down-regulated by at least 2-fold (Figure S1 and Table S1). GO analyses for the up- regulated genes identified GO terms relating to sterol and lipid metabolism, which were significantly enriched in the up-regulated dataset (Figure S1). We were unable to identify any significantly enriched terms in the down-regulated dataset. Compared with Androgen-Dependent Exons previous analyses (for example, see reference [22]), the response to androgens in this current study is thus typical of AR-regulated gene expression (Figure S1). Approximately half of the androgen–responsive genes identified in our analysis have been previously found to be androgen- regulated (Table S1) in at least one of seven previously published datasets [16,17,18,19,20,21,22]. We did, however, additionally identify a subset of 226 novel genes transcriptionally regulated in response to androgens (Table S1) including LYN, which encodes a tyrosine kinase implicated in PCa cell biology [23]. Western analysis of lysates made from LNCaP cells grown in steroid- deplete medium and following 24 hours androgen treatment confirmed that the fall in LYN mRNA expression equated to a fall in LYN protein expression (Figure 1A). We next used the IPA ‘‘Core Analysis’’ function to identify clusters of novel androgen- regulated genes associated with, and including the tyrosine kinase LYN (Figure 1B). Using RT-PCR, we confirmed statistically- significant up-/down-regulation of LYN and eleven LYN-associated genes relative to GAPDH expression in each of the treatment samples as compared with controls (p,0.05) exactly as predicted by our microarray analysis (Figure 1B). The validation rate (100%) suggests that our microarray analysis is a robust description of androgen-dependent transcriptional changes in PCa cells. To detect changes in mRNA isoform ratios in response to androgen stimulation we applied PLIER/Iter-PLIER statistical analysis to exon level data along with several filtering criteria (see Materials and Methods). We identified a total of 1279 differentially expressed probesets (p,0.001), of which 325 (,25%) mapped to well known alternative splicing events (n = 108; 8.4%), alternative first exons/59 ends (n = 144; 11.3%) and alternative last exons/39 ends (n = 73; 5.7%). The remaining probesets did not have obvious annotations. In view of previously-published data [14,15], we focussed solely on cassette exons and changes in extreme 59-ends of transcripts (alternative transcriptional initiation sites due to alternative promoter selection) for which there were full length annotated UCSC (University of California, Santa Cruz) gene sequences [24] available based on RefSeq or GenBank entries with known exon connectivity. Probe intensities of candidate transcripts were visualised at single exon level using EASANA to select candidates that might encode potentially androgen-regulated alternative mRNA isoforms for experimental verification. We identified a total of 30 candidate mRNA isoforms, of which 7 were positively validated in the array sample set as well as an independent dataset (Figures 2 and 3, and fully described below). In each validated case we found statistically significant differences (p,0.05) in actual biological ratios of constitutive and alternative (androgen-regulated) mRNA isoforms as monitored by multiplex RT-PCR and fluorescent quantification after capillary agarose gel electrophoresis (Figure 3). In each case, RT-PCR products were sequenced to verify the content of individual mRNA species. Identification of mRNAs containing androgen-regulated cassette exons There are two fully annotated mRNA isoforms from the NDUFV3 gene (RefSeq accession numbers NM_021075 and NM_001001503), which encodes a subunit of the NADH- ubiquinone oxidoreductase complex. Our analysis suggested differential androgen-regulated production of these two isoforms, with androgen-dependent skipping of a cassette exon (exon 3) within the mRNA transcript. Although no direct function for this gene product has been described in PCa, mitochondrial complex I, of which the NDUFV3 protein is a component, plays an important role in interferon-beta and retinoic acid-induced cancer cell death. The long (1095 nt; nucleotide) cassette exon within the NDUFV3 PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29088