RNase L dimerization in a mammalian two-hybrid system in response to 2',5'-oligoadenylates.
RNase L, a key enzyme in the anti-viral activity of interferons, requires 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 measured with a mammalian two-hybrid system. Hybrid proteins consisting of the GAL4 DNA binding domain fused to RNase L and the VP16 transactivation domain fused to RNase L were able to associate and drive transcription of a reporter gene, but only after cells were transfected with 2-5A. Several functional forms of 2-5A, such as p3A2'p5'A2'p5'A, were capable of activating transcription in human HeLa cells. In contrast, p3A2'p5'A, which can neither activate nor dimerize RNase L, did not induce 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-throughput screening method for RNase L activators which could lead to the discovery of novel anti-viral and anti-cancer agents.
Naik
S
S
Department of Cancer Biology, NN1-06, The Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
Paranjape
J M
JM
Silverman
R H
RH
D000227:Adenine Nucleotides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D009843:Oligoribonucleotides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010449:Peptide Mapping; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D000227:Adenine Nucleotides; D017931:DNA Primers; D009843:Oligoribonucleotides; D011993:Recombinant Fusion Proteins; C023505:2',5'-oligoadenylate; D008156:Luciferases; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease; D001616:beta-Galactosidase