2-5A dependent RNase (RNase L, RNase F) is the terminal factor in an interferon-regulated, RNA degradation pathway which has the potential to inhibit both viral and cellular growth. We have cloned and expressed a cDNA which contains the coding sequence of 2-5A dependent RNase. The goals of this project are to perform incisive molecular studies on 2-5A dependent RNase and its gene and to determine the biological significance of the 2-5A system. To obtain basic information about the structure and function of 2-5A dependent RNase, molecular analysis will be performed on the complete protein purified from recombinant cells. To identify the 2-5A binding site this domain will be tagged with a photoaffinity probe, isolated and sequenced. Synthetic peptides and mutant versions of 2-5A dependent RNase will permit identification of which amino acid residues are involved in 2-5A binding and in the inhibition of 2-5A binding after phosphorylation by protein kinase C. To study events controlling 2-5A dependent RNase levels we will monitor transcription during interferon-treatment, growth arrest, and cell differentiation. In addition, the promoter of the 2-5A dependent RNase gene will be isolated, sequenced, and analyzed both for binding to specific nuclear factors and for regulated expression after fusion to a reporter gene. To study the role of the 2-5A system in interferon action we will express sense and antisense RNA for both 2-5A dependent RNase and 2-5A synthetase in mammalian cells. Effects of enhancing or diminishing levels of these enzymes on virus and cell growth will be determined. To downregulate 2-5A dependent RNase levels more efficiently, ribozymes which will specifically cleave mRNA for 2-5A dependent RNase will be expressed in cells allowing effects on interferon action, cell growth, and differentiation to be determined. To establish if mRNA species containing an AU-rich sequence are preferentially cleaved by 2-5A dependent RNase, we will measure breakdown of specific mRNA species in recombinant cells containing enhanced or diminished levels of 2-5A dependent RNase. Because the 2-5A system has the ability to inhibit both virus replication and neoplastic cell growth this project directly impacts on the control of virus infection and cancer in man.
Showing the most recent 10 out of 103 publications
