Recurrent chimeric RNAs enriched in human prostate cancer identified by deep sequencing

Abstract

Transcription-induced chimeric RNAs, possessing sequences from different genes, are expected to increase the proteomic diversity through chimeric proteins or altered regulation. Despite their importance, few studies have focused on chimeric RNAs especially regarding their presence/roles in human cancers. By deep sequencing the transcriptome of 20 human prostate cancer and 10 matched benign prostate tissues, we obtained 1.3 billion sequence reads, which led to the identification of 2,369 chimeric RNA candidates. Chimeric RNAs occurred in significantly higher frequency in cancer than in matched benign samples. Experimental investigation of a selected 46 set led to the confirmation of 32 chimeric RNAs, of which 27 were highly recurrent and previously undescribed in prostate cancer. Importantly, a subset of these chimeras was present in prostate cancer cell lines, but not detectable in primary human prostate epithelium cells, implying their associations with cancer. These chimeras contain discernable 5' and 3' splice sites at the RNA junction, indicating that their formation is mediated by splicing. Their presence is also largely independent of the expression of parental genes, suggesting that other factors are involved in their production and regulation. One chimera, TMEM79-SMG5, is highly differentially expressed in human cancer samples and therefore a potential biomarker. The prevalence of chimeric RNAs may allow the limited number of human genes to encode a substantially larger number of RNAs and proteins, forming an additional layer of cellular complexity. Together, our results suggest that chimeric RNAs are widespread, and increased chimeric RNA events could represent a unique class of molecular alteration in cancer.

Fig. 1.

Strategy for identification and validation of chimeric RNAs. Paired reads are mapped to both the genome and the transcriptome, and if each of the paired reads maps to different genes, then it is considered to be a paired “chimeric” read representative of a putative chimeric event. On the basis of the alignment and location of paired chimeric reads, primers (red arrows) are designed to amplify the putative RNA junction from patients’ RNAs. RT-PCR and Sanger sequencing lead to the identification of the RNA junction of the chimera. Using the RNA junction as template, previously unmappable reads are aligned to the template and thus “junction” reads are identified. These junction reads have one read aligning to the RNA junction and the other read to one of the genes.

Fig. 2.

Global analysis of chimeric RNAs. (A) A total of 2,369 putative chimeric events were identified on the basis of stringent bioinformatic criteria described in SI Materials and Methods. The frequency of distribution based on reads from all samples for all 2,369 distinct chimeric events is shown. Events chosen for experimental validation are shown in purple. (B) Comparison of the average number of paired chimeric reads showed that chimeric reads are significantly more abundant in cancer samples than in matched benign samples.

Fig. 3.

Chimeric RNAs are highly recurrent in prostate cancer. The relative frequency of recurrence of chimeric RNAs in each cancer and matched benign sample is shown. Each vertical column represents data from one patient sample and each horizontal row represents relative abundance for a particular chimeric event in each patient sample. Yellow indicates high occurrence and black low occurrence. C, cancer sample; N, matched benign samples. The white line separates cancer from matched benign samples. The P value obtained by the Kolmogorov–Smirnov test for each chimeric RNA is shown on the right. All data are compared at the same scale except TMPRSS2-ERG, which is normalized to a different scale so that its relative expression level can be compared between samples without color saturation.

Fig. 4.

Examples of chimeric RNA and parental gene expression in additional patient cohorts and prostate cancer cell lines. (A) Results of RT-PCR analysis for the indicated chimeric RNAs and their parental genes in 10 cancer samples (C41–C50) and 5 noncancer donor samples (D1–D5). TMEM79-SMG5 is undetectable in the donors without cancer whereas the parental genes SMG5 and TMEM79 are expressed. (B) Results of RT-PCR analysis for the indicated chimeric RNAs in prostate cell lines PrEC, PNT1a, LNCaP, LAPC4, VCaP, DU145, and PC3. This subset of chimeric RNAs is undetectable in PrEC. As expected, TMPRSS2-ERG is highly expressed in VCaP, whereas the control actin is expressed in all cell lines. (C) Expression of parental genes in PrEC for those chimeric RNAs shown in B. Although this subset of chimeric RNAs is not expressed in PrEC, the expression of many parental genes is clearly detected.

Fig. 5.

Schematic of TMEM79-SMG5 chimeric RNA in prostate cancer. Chimeric RNAs can result in a new 5′-UTR and truncated ORF as in the case of TMEM79-SMG5. In the pictogram, coding exons are represented by blocks connected by horizontal lines representing introns. The 5′- and 3′-UTRs are represented by shorter blocks. Arrows indicate direction of transcription of parental genes. Dashed lines indicate the boundaries of the sequence contributing to chimeric RNAs. Splice junctions are shown as uppercase letters. The sequences in black are part of the introns removed in the formation of the chimeric RNAs.

Fig. 6.

The TMEM79-SMG5 chimera as a potential diagnostic marker. (A) The relative expression level of the TMEM79-SMG5 chimera (normalized to GAPDH) in PrEC, 18 donors without cancer, and 54 patients with prostate cancer, determined by quantitative RT-PCR. These additional samples were not used for high-throughput sequencing. (B) Box plot showing the range of relative expression levels in patients with prostate cancer vs. donors without cancer. TMEM79-SMG5 was expressed at a significantly high level in patients with prostate cancer (P value = 1.048 e-05). Sample minimum and maximum are represented by horizontal lines. The box represents the lower and upper quartile separated by a thick line, which is the median. The circles represent values that are considered to be outliers.