Jiang Yan, Price David H
Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA.
Cell Cycle. 2004 Sep;3(9):1151-3. Epub 2004 Sep 10.
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.
TTF2是一种RNA聚合酶II终止因子，负责有丝分裂期转录延伸的抑制。正常情况下，所有RNA聚合酶II分子都会从浓缩的有丝分裂染色体上消除，但小干扰RNA（siRNA）介导的TTF2敲低会导致一种表型，即聚合酶保留在有丝分裂染色体上。为了证明这种表型是由于TTF2活性降低所致，我们构建了一种抗siRNA的TTF2替代载体，该载体表达一种编码绿色荧光蛋白（GFP）标记的TTF2的mRNA，该mRNA在siRNA靶向区域含有沉默突变。瞬时转染实验表明，内源性TTF2和GFP标记的野生型TTF2对siRNA均敏感，但含有错配的cDNA编码的GFP标记的TTF2在存在TTF2-siRNA的情况下大量表达。重要的是，抗siRNA的GFP标记的TTF2的表达挽救了TTF2-siRNA所见的有丝分裂表型，证明TTF2减少是RNA聚合酶II保留在有丝分裂染色体上的原因。
