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. 2018 Dec 11;115(52):E12295–E12304. doi: 10.1073/pnas.1814704115

Fig. 4.

Fig. 4.

RNA-mediated interchromosomal gene fusion between TMPRSS2 and ETV1. (A) RT-PCR shows that only the antisense RNA targeting stem TETV-1 (with the highest stem Tm = 72 °C) led to induced fusion transcript (lane 1). Antisense RNAs targeting stems with lower genomic DNA stem stabilities (TETV-2 to -8, lanes 2–8) resulted in no detectable induction. (B) The corresponding sense input RNA targeting the same TETV-1 stem failed to induce fusion transcript (lane 1 vs. lane 2). (C) Gene fusion is specified by the sequence of input RNA used. The antisense TETV-1 induced TMPRSS2–ETV1 fusion but not TMPRSS2–ERG fusion (lane 2). Conversely, antisense-5 induced TMPRSS2–ERG fusion but not TMPRSS2–ETV1 fusion (lane 1). (D) RT-PCR shows the transient nature of input RNA which was present at day 3 but not day 47 postinitial treatment (Lower, lane 1 vs. lane 2), and the persistent nature of the induced fusion transcript (Upper) up to 47 d postinitial treatment in the enriched LNCaP population. (E, Upper) Schematics of three identified genomic breakpoints marked as x, y, and z, and the primers used to amplify the breakpoints. (Lower) The unrearranged wild-type TMPRSS2 allele was revealed by primer pair E/F (990 bp; lanes 1 and 4) and G/H (956 bp; lanes 7 and 10), and the unrearranged wild-type ETV1 allele by primer pair M/N (1,024 bp; lanes 2, 5, 8, and 11). The genomic fusion band x (1,150 bp) and y (1,044 bp) amplified by fusion-specific primer pair E/N, and fusion band z (1,043 bp) amplified by primer pair G/N, were present only in the induced and enriched LNCaP population but absent in untransfected LNCaP cells (lane 6 vs. lane 3, and lane 9 vs. lane 12). (F) Sanger sequencing of the x, y, and z fusion band identified the exact genomic breakpoints. Region of microhomology at the breakpoints are boxed by solid lines, and indels by dash lines. The full-length Sanger sequences are shown in SI Appendix, Figs. S22–S24. (G) FISH probes against TMPRSS2 gene (red) on chromosome 21 and against ETV1 gene (green) on chromosome 7 were used to confirm the gene fusion. Examples of FISH signal in an untransfected cell (i) and a cell carrying induced TMPRSS2–ETV1 gene fusion (ii) are shown. Arrow points to the colocalized FISH signals indicative of TMPRSS2–ETV1 gene fusion, which is shown at a higher magnification in the inlet. About 0.9% of the enriched population (30 of 3,301 cells) was positive for TMPRSS2–ETV1 fusion gene based on the colocalized FISH signals. In contrast, none of the cells from the untransfected population (0 of 620 cells) showed colocalized FISH signals.