The self-cleaving domain from the genomic RNA of hepatitis delta virus: sequence requirements and the effects of denaturant

Abstract

The sequence requirements for self-cleavage of hepatitis delta virus genomic RNA were examined using precursor RNAs which were labeled at either the 5' or 3' ends and progressively deleted from the unlabeled end. In the presence of 50% formamide, which enhances self-cleavage in 2 mM MgCl2 at 37 degrees C, 84 nucleotides (nt) 3' of the break site were required. In the absence of formamide the minimum was reduced to 82 nt. Under both sets of conditions, precursors with 1 nt 5' to the break site cleaved. These results allowed two condition-dependent minimal domains for self-cleavage to be defined. However, in the absence of formamide, sequences flanking the minimal domain inhibited cleavage, possibly through involvement in the formation of non-cleaving structures. These data are consistent with the idea that cleavage in vivo could be regulated by alternative RNA structures.