RNASE-L DIMERIZATION IN A MAMMALIAN 2-HYBRID SYSTEM IN RESPONSE TO 2',5'-OLIGOADENYLATES

RNase L, a key enzyme in the anti-viral activity of interferons, requi res activation by 2',5'-linked oligoadenylates (2-5A) to cleave viral and cellular single-stranded RNA. Here we demonstrate that 2-5A causes formation of stable dimers of RNase L in intact human cells as measur ed with a mammalian two-hybrid system, Hybrid proteins consisting of t he GAL4 DNA binding domain fused to RNase L and the VP16 transactivati on domain fused to RNase L were able to associate and drive transcript ion of a reporter gene, but only after cells were transfected with 2-5 A. Several functional forms of 2-5A, such as p(3)A2'p5'A2'p5'A, were c apable of activating transcription in human HeLa cells. In contrast, p (3)A2'p5'A, which can neither activate nor dimerize RNase L, did not i nduce gene expression. Evidence for the involvement of the C-terminal region of RNase L in dimerization was obtained by expressing truncated forms of RNase L. These findings describe a convenient, high-throughp ut screening method for RNase L activators which could lead to the dis covery of novel anti-viral and anti-cancer agents.