A variant of the KLK4 gene is expressed as a cis sense-antisense chimeric transcript in prostate cancer cells

Abstract

In humans, more than 30,000 chimeric transcripts originating from 23,686 genes have been identified. The mechanisms and association of chimeric transcripts arising from chromosomal rearrangements with cancer are well established, but much remains unknown regarding the biogenesis and importance of other chimeric transcripts that arise from nongenomic alterations. Recently, a SLC45A3-ELK4 chimera has been shown to be androgen-regulated, and is overexpressed in metastatic or high-grade prostate tumors relative to local prostate cancers. Here, we characterize the expression of a KLK4 cis sense-antisense chimeric transcript, and show other examples in prostate cancer. Using non-protein-coding microarray analyses, we initially identified an androgen-regulated antisense transcript within the 3' untranslated region of the KLK4 gene in LNCaP cells. The KLK4 cis-NAT was validated by strand-specific linker-mediated RT-PCR and Northern blotting. Characterization of the KLK4 cis-NAT by 5' and 3' rapid amplification of cDNA ends (RACE) revealed that this transcript forms multiple fusions with the KLK4 sense transcript. Lack of KLK4 antisense promoter activity using reporter assays suggests that these transcripts are unlikely to arise from a trans-splicing mechanism. 5' RACE and analyses of deep sequencing data from LNCaP cells treated +/-androgens revealed six high-confidence sense-antisense chimeras of which three were supported by the cDNA databases. In this study, we have shown complex gene expression at the KLK4 locus that might be a hallmark of cis sense-antisense chimeric transcription.

FIGURE 1.

Validation of the KLK4 antisense transcript. (A) A microarray probe (red box with white arrowhead) detected a 3.38-fold androgen up-regulated KLK4 antisense transcript (pink box) in LNCaP prostate cancer cells. Primers used in subsequent figures are shown relative to the classical KLK4 sense transcript, which is encoded by five exons (blue boxes). Dotted lines represent unknown antisense sequences. (B) Diagram of the RT-PCR approach taken to differentiate overlapping sense antisense transcripts. (C) Validation of the KLK4 sense and antisense transcripts using strand-specific primers conjugated to synthetic linker sequences. PCR was carried out at 30 cycles using linker 1 (L1) and linker 2 (L2) primers, and the specified KLK4 primer.

FIGURE 2.

Mapping of the KLK4 antisense transcription start site (TSS) in LNCaP cells. (A) Two transcripts were initially identified for the KLK4 antisense transcript by 5′ RLM-RACE. (B) Exon usage of the sense–antisense chimeric transcripts identified by 5′ RLM-RACE. Also shown is the location and direction of the internal primer (Ex5S-2) used in 5′ RACE. (C) BLAT results of other sense–antisense variants identified by 5′ RACE.

FIGURE 3.

Mapping of the 3′ end of the KLK4 antisense transcript in LNCaP cells. (A) One transcript of 778 bp was initially detected by 3′ RACE. (B) Diagram of the exon and intron usage of the 3′-RACE product. The arrow represents the location and direction of the internal KLK4 primer (Ex5AS-1) used in 3′ RACE. (D) BLAT results of other sense–antisense chimeras identified in 3′ RACE. (E) Northern blot targeting the antisense transcript detects an ∼1.5-kb transcript in cells treated with androgens (R1881) but not in mock-treated cells (ethanol). (F) Diagram showing the potential exon (numbered boxes) and intron (Int2) usage of a transcript comprising both 5′- and 3′-RACE sequences.

FIGURE 4.

Further analysis of gene transcription at the KLK4 locus. Shown are the BLAT results from 3′ RACE using primers targeting the sense transcript. The three more readily detected transcripts include the classical KLK4 transcript, a premature intron 4 truncated transcript that is already in the cDNA databases, and a KLK4–KLP1 chimera.

FIGURE 5.

Assessing for basal promoter activity of the KLK4 antisense transcript for evidence of independent KLK4 antisense transcription. (A) Diagram showing retrotransposon elements (SINE and LINE) and a cluster of predicted sp1 and transcription start sites (TSS) located within the putative antisense promoter. Also shown are the locations of the three inserts used in luciferase reporter assays. The nucleic acid positions are relative to the reverse primer used to generate the promoter constructs. (B) Assessing basal promoter activity of the antisense promoter using reporter assays. Data are represented as SEM from three independent experiments that were each carried out in quadruplicate. Data were normalized to pGL3-Basic activity.

FIGURE 6.

Genome-wide identification of sense–antisense chimeric splice events. (A) Identification of a cis sense–antisense transcript in another MSR1 gene (ISOC2) gene by 5′ RACE. (B) Frequency distribution of LNCaP mock- (left) and DHT- (right) treated RNA-seq tags mapping across sense–antisense chimeric exon–exon junctions. (Red bars) Frequencies of sense–antisense chimeric exon–exon junctions (RefSeq exon paired with antisense mRNA/EST exon within 10 kb); (blue bars) frequencies of background chimeric exon–exon junction mappings (RefSeq exon paired with random mRNA/EST exon). Informative tags (indicated) were defined within regions where no background mapping (i.e., mapping by chance) occurred. (C) Three genes (dark blue) identified from RNA-seq analysis as sharing exon–exon junctions with antisense transcripts (light blue) with corroborating cDNA transcripts (black). Transcriptional direction and splice junctions are indicated.