Androgen-Dependent Exons Figure 1. Expression of LYN and -associated genes are androgen-regulated in LNCaP cells. (A) Western analysis of LYN and actin (loading control) protein expression in cells grown in full medium, steroid-deplete medium, and following 24 hours of treatment with 10 nM R1881. (B) Ingenuity Pathway Analysis (IPA) of LYN-associated genes. Novel androgen-regulated genes identified in the present study are labelled in shades of red (up-regulated genes) or shades of green (down-regulated genes). (C) Expression of 12 novel androgen-regulated genes associated with LYN in steroid-deplete conditions (2, lanes 1–4) and after 24 hours of treatment with 10 nM R1881 (+, lanes 5–8) measured by multiplex RT-PCR. Levels of transcript expression were normalised to GAPDH levels, and data from experimental quadruplicates were used to obtain the mean 6 SE for each transcript. A particularly high level of androgen-mediated gene activation was seen for the FZD5 gene, which encodes a transmembrane Wnt5a receptor. Other confirmed up-regulated genes included KITLG, which encodes the Kit ligand upstream of LYN in the pathway, and MTOR, which encodes a key regulator of cell growth. Similarly, we confirmed down-regulation of the novel androgen-regulated genes REDD1 (also known as DDIT4), which negatively regulates mTOR, and DEPDC6 which encodes an mTOR interacting protein. doi:10.1371/journal.pone.0029088.g001 transcript inserts coding information for a 365 amino acid (aa) peptide into the NDUFV3 protein sequence compared with the shorter mRNA isoform (Figure 2, NCBI NP_066553 and NP_001001503.1 respectively). Multiplex RT-PCR demonstrated expression of exon 3-included and exon 3-skipped transcripts in steroid-deplete conditions, and confirmed an androgen-dependent switch in exon 3 inclusion (Figure 3A, compare lanes 1–4 with 5– 8). Following androgen treatment there was a ,2 fold increase in the exon-skipped product relative to the exon-included product (Figure 3B). Androgen-regulated inclusion of a cassette exon was also identified in the transcript derived from the gene encoding the putative transcription factor ZNF121. ZNF121 encodes a 390 amino acid protein with 9 C2H2-type zinc finger motifs (Figure 2C), and is expressed as three alternative mRNA isoforms (UCSC mRNA isoforms uc010xkp.1, uc010dwt.2 and uc010xkq.1, respectively). Exon 2 is alternatively-spliced within the 59 untranslated region (UTR), while inclusion of exon 5 interrupts the reading frame initiated in exon 4, and results in a frameshifted mRNA (Figure 2A and 3B). Using multiplex RT- PCR, exon 2 was found to be constitutively skipped in LNCaP cells (data not shown), but inclusion of the 58 nt exon 5 was significantly up-regulated in response to androgen treatment (Figure 3A, compare lanes 1–4 with lanes 5–8). In terms of functional outputs, cassette exon 5 introduces a frameshift to the ZNF121 protein sequence very close to the amine (N)-terminus of Figure 2. Overview of identified androgen-regulated mRNA isoforms. (A) Cartoon of intron/exon structure of gene, showing the splicing pattern and the positions of the primers (shown as arrows) used for RT-PCR. Genes are annotated with the androgen responsive splicing/promoter selection event above the gene (exons joined by broken lines in black), and constitutive event below the gene (exons joined by broken lines in grey). The androgen-regulated exons are shown in black, and weakly androgen-dependent exons indicated in grey. Constitutive exons are indicated in white. The positions of the primers used for RT-PCR validation are shown below exons as arrows, and predicted position of alternative promoters are shown as angled arrow. (B) For each gene a pair of alternative mRNA transcript isoforms is shown. (C) The predicted full-length proteins made from each of the genes are indicated, along with the position of conserved domains. These are abbreviated: ZF = zinc finger motif; HDc = metal dependent phosphohydroxylate with conserved HD motif; PolIII = DNA polymerase subunits gamma and tau; CC = coiled coil domain; Tub = tuberin superfamily; Rap/GAP = RAP/GTPase Activating Protein superfamily; PDZ = protein interaction and binding domain; C2 = protein kinase C conserved domain 2 Ca2+ binding motif; STYKc = STYK protein kinase domain. doi:10.1371/journal.pone.0029088.g002 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29088