Fig. 5.

(A) RNA-mediated gene fusion requires RNA/DNA hybrid formation. Plasmids expressing input RNAs (antisense-5 for TMPRSS2–ERG, and antisense TETV-1 for TMPRSS2–ETV1) were cotransfected in ratio of 2:3 with plasmids expressing either wild-type or mutant RNaseH. The corresponding sense input RNAs were also cotransfected with wild-type or mutant RNaseH as the controls. (Top and Middle) Induction of fusion gene by antisense input RNA was significantly reduced in the presence of wild-type RNaseH vs. the mutant RNaseH (TMPRSS2–ERG: lane 2 vs. lane 1; TMPRSS2–ETV1: lane 6 vs. lane 5). Sense input RNAs failed to induce fusion regardless of the expression of RNaseH (lanes 3, 4, 7, and 8). (Bottom) RT-PCR showed that equal amount of wild-type or mutant RNase H was expressed. (B) The disparity between antisense versus sense input RNA is due to transcriptional conflict. The input RNAs were expressed by U6 (a pol-III promoter) while α-amanitin was used to inhibit pol-II transcription of the parental genes for various time periods (0, 2, 6, 12, and 24 h). α-amanitin was then rinsed off so that the newly induced fusion gene can express the fusion RNA. The induced fusion RNA was assayed by RT-PCR at day 3. Antisense-5 and sense-5 are for inducing TMPRSS2–ERG (Upper); antisense TETV-1 and sense TETV-1 are for inducing TMPRSS2–ETV1 (Lower). The sense input RNAs that previously failed to induce fusion, began to induce TMPRSS2–ERG (lanes 9 and 10) after 12 h of α-amanitin treatment, and TMPRSS2–ETV1 fusion (lane 20) after 24 h of α-amanitin treatment, respectively. GAPDH is used as internal loading control.