A Sequence Element Necessary for Self-Cleavage of the Antigenomic Hepatitis Delta RNA in 20 M Formamide

Abstract

The genomic and antigenomic RNAs of hepatitis delta virus are capable of self-cleavage and show no significant sequence similarities to other known self-cleaving RNAs. We have derived an antigenomic delta RNA which cleaves to completion in 15 s in 9 mM magnesium at 37 °C and is capable of efficient self-cleavage in concentrations of formamide as high as 20 M. Cleavage in high concentrations of denaturant is dependent upon the presence of a poly purine sequence element, GGAGA, located between 81 and 85 nucleotides downstream of the cleavage site. Mutation of the initial G81G82to C81C82, or removal of the sequence element, results in a loss of the ability to cleave in high formamide concentrations. Changing the final U-2C-1, of a pyrimidine-rich region, UCUUC, just upstream of the cleavage site, to G-2G-1severely affects the self-cleavage, but introducing the two mutations, GG to CC and UC to GG, into the same molecule, restoring potential base pairing, partially restores the formamide stability. Relocating the GGAGA sequence upstream of the cleavage site also results in partial restoration of the formamide cleavage. Although the GGAGA sequence is important for self-cleavage under denaturing conditions, it does not appear to be necessary for HDV RNA cleavage in normal buffer conditions. © 1992, American Chemical Society. All rights reserved.