Nucleotide Sequence Requirements for Self-cleavage of Neurospora VS RNA

Abstract

We have used several complementary approaches to investigate the minimal contiguous sequence required for the in vitro self cleavage reaction performed by Neurospora VS RNA. Deletion analysis and site-directed mutagenesis revealed that only a single nucleotide is required upstream of the self-cleavage site, and that the identity of this nucleotide is not critical. This distinguishes VS RNA from all currently known ribozymes except hepatitis delta virus RNA. The shortest contiguous sequence capable of cleavage contains 153 nt downstream of the cleavage site. Linker insertion mutagenesis suggests that much of this downstream sequence is important for self-cleavage. Comparative sequence analysis of the VS plasmid from six natural isolates supports the importance in vivo of the minimal region determined by in vitro methods. Also, phylogenetic analysis raises the possibility of a recent horizontal transfer of the VS plasmid from Neurospora intermedia to Neurospora sitophila.