Determination of the Role of the Human RNase H1 in the Pharmacology of DNA-like Antisense Drugs

摘要

Although ribonuclease H activity has long been implicated as a molecular mechanism by which DNA-like oligonucleotides induce degradation of target RNAs, definitive proof that one or more RNase H is responsible is lacking. To date, two RNase H enzymes (H1 and H2) have been cloned and shown to be expressed in human cells and tissues. To determine the role of RNase H1 in the mechanism of action of DNA-like antisense drugs, we varied the levels of the enzyme in human cells and mouse liver and determined the correlation of those levels with the effects of a number of DNA-like antisense drugs. Our results demonstrate that in human cells RNase H1 is responsible for most of the activity of DNA-like antisense drugs. Further, we show that there are several additional previously undescribed RNases H in human cells that may participate in the effects of DNA-like antisense oligonucleotides. Although ribonuclease H activity has long been implicated as a molecular mechanism by which DNA-like oligonucleotides induce degradation of target RNAs, definitive proof that one or more RNase H is responsible is lacking. To date, two RNase H enzymes (H1 and H2) have been cloned and shown to be expressed in human cells and tissues. To determine the role of RNase H1 in the mechanism of action of DNA-like antisense drugs, we varied the levels of the enzyme in human cells and mouse liver and determined the correlation of those levels with the effects of a number of DNA-like antisense drugs. Our results demonstrate that in human cells RNase H1 is responsible for most of the activity of DNA-like antisense drugs. Further, we show that there are several additional previously undescribed RNases H in human cells that may participate in the effects of DNA-like antisense oligonucleotides. RNase H hydrolyzes RNA in RNA-DNA hybrids (1Stein H. Hausen P. Science. 1969; 166: 393-395Crossref PubMed Scopus (179) Google Scholar). RNase H activity appears to be ubiquitous in eukaryotes and bacteria (2Itaya M. Kondo K. Nucleic Acids Res. 1991; 19: 4443-4449Crossref PubMed Scopus (63) Google Scholar, 3Itaya M. McKelvin D. Chatterjie S.K. Crouch R.J. Mol. Gen. Genet. 1991; 227: 438-445Crossref PubMed Scopus (54) Google Scholar, 4Kanaya S. Itaya M. J. Biol. Chem. 1992; 267: 10184-10192Abstract Full Text PDF PubMed Google Scholar, 5Busen W. J. Biol. Chem. 1980; 255: 9434-9443Abstract Full Text PDF PubMed Google Scholar, 6Rong Y.W. Carl P.L. Biochemistry. 1990; 29: 383-389Crossref PubMed Scopus (34) Google Scholar, 7Eder P.S. Walder J.A. Biochimie (Paris). 1993; 75: 6472-6479Crossref Scopus (103) Google Scholar). Although RNases H constitutes a family of proteins of varying molecular weight, the nucleolytic activity and substrate requirements appear to be similar for the various isotypes. For example, all RNases H studied to date function as endonucleases exhibiting limited sequence specificity and requiring divalent cations (e.g. Mg2+, Mn2+) to produce cleavage products with 5′ phosphate and 3′ hydroxyl termini (8Crouch R.J. Dirksen M.L. Linn S.M. Roberts R.J. Cold Spring Harbor. Cold Spring Harbor Press, Plainview, New York1982: 211-254Google Scholar). Although a number of viral and bacterial polymerases and exonucleases have been shown to have RNase H activities (8Crouch R.J. Dirksen M.L. Linn S.M. Roberts R.J. Cold Spring Harbor. Cold Spring Harbor Press, Plainview, New York1982: 211-254Google Scholar), in mammalian cells only two classes of RNase H enzymes have been identified (5Busen W. J. Biol. Chem. 1980; 255: 9434-9443Abstract Full Text PDF PubMed Google Scholar, 9Eder P.S. Walder J.A. J. Biol. Chem. 1991; 266: 6472-6479Abstract Full Text PDF PubMed Google Scholar, 10Frank P. Albert S. Cazenave C. Toulme J.J. Nucleic Acids Res. 1994; 22: 5247-5254Crossref PubMed Scopus (40) Google Scholar). These enzymes were shown to differ with respect to co-factor requirements and were shown to be inhibited by sulfhydryl reagents (10Frank P. Albert S. Cazenave C. Toulme J.J. Nucleic