Discovery of CTCF-Sensitive Cis-Spliced Fusion RNAs between Adjacent Genes in Human Prostate Cells

Abstract

Genes or their encoded products are not expected to mingle with each other unless in some disease situations. In cancer, a frequent mechanism that can produce gene fusions is chromosomal rearrangement. However, recent discoveries of RNA trans-splicing and cis-splicing between adjacent genes (cis-SAGe) support for other mechanisms in generating fusion RNAs. In our transcriptome analyses of 28 prostate normal and cancer samples, 30% fusion RNAs on average are the transcripts that contain exons belonging to same-strand neighboring genes. These fusion RNAs may be the products of cis-SAGe, which was previously thought to be rare. To validate this finding and to better understand the phenomenon, we used LNCaP, a prostate cell line as a model, and identified 16 additional cis-SAGe events by silencing transcription factor CTCF and paired-end RNA sequencing. About half of the fusions are expressed at a significant level compared to their parental genes. Silencing one of the in-frame fusions resulted in reduced cell motility. Most out-of-frame fusions are likely to function as non-coding RNAs. The majority of the 16 fusions are also detected in other prostate cell lines, as well as in the 14 clinical prostate normal and cancer pairs. By studying the features associated with these fusions, we developed a set of rules: 1) the parental genes are same-strand-neighboring genes; 2) the distance between the genes is within 30kb; 3) the 5′ genes are actively transcribing; and 4) the chimeras tend to have the second-to-last exon in the 5′ genes joined to the second exon in the 3′ genes. We then randomly selected 20 neighboring genes in the genome, and detected four fusion events using these rules in prostate cancer and non-cancerous cells. These results suggest that splicing between neighboring gene transcripts is a rather frequent phenomenon, and it is not a feature unique to cancer cells.

Investigation of fusion gene expression in HCT116 cells

“…As a rule, fusion products in the genome are formed through DNA rearrangement (26)(27)(28). Fusion RNA transcripts in the transcriptome comprised of cis-splicing of adjacent genes and trans-splicing (fusion between genes on different strands of the same chromosome or different chromosomes) (4,7,11,29). Our study revealed that the existence of fusion RNA transcripts was not always accompanied by DNA rearrangements.…”

“…CTCF regulates the expression of fusion RNA transcripts, including SLC45A3-ELK4 and ADCK4-NUMBL chimeric RNA transcripts (7,11). The cis-splicing of adjacent genes was identified to be regulated by the binding events of CTCF to genome.…”

“…As there are ~15,000 binding sites for CTCF in the human genome (10), CTCF may regulate the expression of numerous other fusion genes. Qin et al (11) identified that CTCF regulates the expression of fusion transcripts that are not unique to cancer cells. CTCF is widely expressed in normal tissues, so may not drive the onset and progression of cancer (10).…”

Fusion transcriptome profiling provides insights into alveolar rhabdomyosarcoma

“…Even though some gene fusions and fusion transcripts have been reported to be present in nonneoplastic samples (8)(9)(10), they tend to be expressed at low levels and may be from the precursors of cancer cells. We and others have shown that fusion transcripts can be detected in normal human cell lines (11) and tissues (12)(13)(14)(15) and can be produced by mechanisms other than chromosomal rearrangement.…”

Fusion RNA profiling provides hints on cell of origin of mysterious tumor

“…3 Contradictory to the prevailing view that fusion RNAs are cancer-unique phenomena, we and others have showed that they are widespread in non-cancer cells and tissues. [4][5][6] We coined the phrase "fusion transcriptome," and demonstrated that "fusion RNA profiling" can cluster biological samples into separate groups of different tissues. To put the concept into use, we used alveolar rhabdomyosarcoma as a model.…”