RNase III Cleaves Eukaryotic Preribosomal RNA at a U3 snoRNP-Dependent Site

A yeast gene homologous to bacterial RNase III ( RNT1) encodes a double-strand–specific endoribonuclease essential for ribosome synthesis. Two rRNA processing events are blocked in cells temperature sensitive for RNT1: cleavage at the snoRNA-dependent A0 site in the 5′ ETS and cleavage in the 3′ ETS. Recombinant RNT1 protein accurately cleaves a synthetic 5′ ETS RNA at the A0 site in vitro, in the absence of snoRNA or other factors. A synthetic 3′ ETS substrate is specifically cleaved at a site 21 nt downstream of the 3′ end of 28S rRNA. These observations show that a protein endonuclease collaborates with snoRNAs in eukaryotic rRNA processing and exclude a catalytic role for snoRNAs at certain pre-rRNA cleavage sites.