the encoded protein, and so may either destabilise the mRNA through the NMD pathway [25] or lead to production of a protein lacking zinc finger motifs (Figure 2C). Identification of mRNAs with distinct androgen-regulated 59 ends We also identified variant mRNA isoforms with alternative 59 exons generated in response to androgen treatment from alternative transcriptional initiation sites due to alternative promoter selection. These included mRNA isoforms from the PDE4D gene, which encodes a cAMP-specific phosphodiesterase involved in signalling pathways, and has been recently identified as a proliferation-promoting factor up-regulated in clinical PCa [26]. The PDE4D gene is transcribed using an array of five alternative transcriptional initiation sites, including distinct promoter elements upstream of exon 1 (which is spliced onto exon 2 and then 7 (RefSeq NM_001165899) and upstream of exon 3 which is also directly spliced onto exon 7 (RefSeq NM_006203)) (Figure 3A) [26]. Using multiplex RT-PCR, LNCaP cells were found to initiate PDE4D transcription upstream of exon 3 in steroid-deplete conditions, and following androgen treatment a dramatic switch in transcriptional initiation was made to the promoter upstream of exon 1 (Figures 2 and 3). Both these PDE4D mRNA isoforms contain predicted ORFs for the conserved HDc motif of metal dependent phosphohydrolases (Figure 2C), but have different N- terminal sequences (NCBI NP_001159371.1 and NP_006194.2). A switch between androgen-regulated mRNA isoforms with distinct 59 ends was also identified for the TACC2 gene, which encodes a centrosomal protein. Multiple different TACC2 mRNA isoforms are transcribed from the TACC2 gene, under the control of alternative internal promoters upstream of exons 14, 15, and 18 (Figure 2A) to give different mRNA isoforms with alternative exon contents [27]. Using multiplex RT-PCR using primers comple- mentary to exons 12 (59 end of the gene) and 21 (39 end of the gene), we confirmed significantly increased levels of 39 end of the TACC2 gene as compared with the 59 end following androgen treatment, suggesting androgen-regulated mRNA isoform produc- tion through an alternative internal AR-activated promoter (Figure 3). The use of a promoter upstream of exon 15 produces a variant mRNA isoform called AZU-1, in which the coding information for the coiled-coil domain is preserved (GenBank ID: AF176646.1). Although TACC2 is a non-essential gene and its deletion does not cause cancer in mice, [28,29], up-regulation of AZU-1 has been implicated in advanced breast cancer [28]. We analysed expression of mRNA isoforms containing TACC2 exons 14, 15 and 18 which are the first exons of each of these annotated transcription units, and found that the promoter upstream of exon 15 is androgen-responsive, indicating AZU-1 is an androgen- regulated transcription unit (data not shown). Androgen-regulated selection of alternative 59 ends were also detected in transcripts derived from the TSC2, RIMS1 (regulating synaptic membrane endocytosis 1) and WEE1 genes. TSC2 encodes an important tumour suppressor protein tuberin, which regulates cell growth [30,31]. The TSC2 gene is complex and encodes a number of different mRNA isoforms with alternative exons, and our analysis predicted increased levels of transcription of the downstream region of the gene. Confirming this by multiplex RT-PCR using primers within exons 8 and 10 (59 end) and 41 and 42 (39 end), we observed an ,2 fold increase in expression of 39 end of the TSC2 gene as compared with the 59 end following androgen treatment (Figure 3A, compare lanes 1–4 with lanes 5– 8). Known annotated mRNA isoforms on UCSC [24,32] indicated two known alternative internal promoters upstream of TSC2 exon 33 (Figure 2A), which might to respond to androgen stimulation Androgen-Dependent Exons (DP1 and DP2). The alternative TSC2 transcript produced in response to androgen treatment contains coding information for the Rap/GAP domain of the full-length protein, but is missing the tuberin family domain (Figure 2C) (GenBank BAG52831.1). Androgen-regulated mRNA isoforms were also predicted within the RIMS1 gene, which is a member of the RAS gene superfamily, and encodes a protein that acts as a scaffold to regulate vesicle exocytosis. UCSC gene annotations [24,32] indicate that RIMS1 encodes a family of 11 different mRNA isoforms, transcribed downstream of 5 promoters. By multiplex RT-PCR using primers within exons 8 and 9 (59 end) and exon 34 and 35 (39 end), we observed an ,3 fold increase in expression of 39 end of the RIMS1 gene as compared with the 59 end following androgen treatment (Figure 3A compare lanes 1–4 with 5–8, and Figure 3B), most likely generated from transcriptional initiation from the UCSC annotated promoters upstream of exon 13 (Figure 2A) [24,32]. The alternative transcripts produced in response to androgen treatment contain a predicted ORF, which excludes a PDZ domain involved in protein interactions and binding, and one calcium-binding (C2, protein kinase C conserved domain 2) domain (Figure 2C) compared with the full length protein. In the above examples, androgen treatment led to alternative promoter selection and an increase in expression of an alternative mRNAs from a particular transcription start site. In contrast, we observed more subtle selective androgen-dependent alternative promoter usage within the androgen down-regulated WEE1 gene. WEE1 encodes an important nuclear tyrosine kinase, which functions as a negative regulator of cell cycle progression and inhibits Cdc2-Cyclin B activity and entry into mitosis. UCSC annotations and published analyses of mRNA isoforms [24,32,33] indicate that WEE1 encodes two alternative mRNAs initiating upstream of exon 1 (isoform 1 encoding full length Wee1 protein) or exon 2 (isoform 2 encoding a shorter protein Wee1i with an alternative N-terminus) (Figure 2). Both the long and the shorter forms of WEE1 protein contain the STY protein kinase domain (STYKc), but the shorter WEE1i does not contain the 214 aa N- terminus domain which mediates ubiquitin-dependent degrada- tion of WEE1 protein to trigger mitotic entry [33]. Multiplex RT- PCR indicated that both WEE1-derived transcripts were down- regulated following androgen treatment, however the longer mRNA isoform (RefSeq NM_003390) was down-regulated to a greater extent as compared with the shorter mRNA isoform (RefSeq NM_001143976) (Figure 3A, compare lanes 1–4 with 5– 8). The relative (,3 fold) increase in abundance of the shorter mRNA isoform (and possibly Wee1i protein) following androgen treatment may provide the cell with constitutive WEE1-like kinase activity (protein NCBI NP_001137448.1) to regulate androgen- induced cell cycle progression (Figure 3B). Novel mRNA isoforms are regulated by endogenous androgens and expressed in benign prostate epithelial cells as well as clinical PCa We validated the above findings using a natural androgen (dihydrotestosterone; DHT) at physiological concentrations (1 nM and 10 nM), as well R1881 at 1 nM concentration, which is thought to positively affect cell proliferation [21] (Figure S2). RT-PCR was performed using total RNA extracted from LNCaP cells grown in steroid-deplete medium and following 24 hours of treatment with different concentrations of R1881 or DHT. The absolute magnitude of the actual biological ratios of constitutive and alternative (androgen-regulated) mRNA isoforms varied for each transcript depending on the dose of R1881 and DHT. Although expression of the alternative mRNA isoform encoded by WEE1 was not affected by 1 nM R1881, differential expression was observed PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29088