Rescue of the TTF2 knockdown phenotype with an siRNA-resistant replacement vector

TTF2 is an RNA polymerase II termination factor that is responsible for mitotic repression of transcription elongation. Normally, all RNA polymerase II molecules are eliminated from condensed mitotic chromosomes, but siRNA-mediated knockdown of TTF2 causes a phenotype in which polymerases are retained on mitotic chromosomes. To prove that this phenotype is due to reduced TTF2 activity we have created an siRNA-resistant TTF2 replacement vector that expresses an mRNA encoding GFP-tagged TTF2 that contains silent mutations in the region that is targeted by the siRNA. Transient transfection experiments demonstrate that endogenous TTF2 and GFP-tagged wild-type TTF2 are both sensitive to the siRNA, but GFP-tagged TTF2 encoded by the cDNA containing mismatches is abundantly expressed in the presence of TTF2-siRNA. Importantly, the mitotic phenotype seen with TTF2-siRNA is rescued by expression of the siRNA-resistant GFP-tagged TTF2 proving that reduced TTF2 is responsible for the retention of RNA polymerase II on mitotic chromosomes.