Rcl1 protein, a novel nuclease for 18 S ribosomal RNA production

Abstract

In all forms of life, rRNAs for the small and large ribosomal subunit are co-transcribed as a single transcript. Although this ensures the equimolar production of rRNAs, it requires the endonucleolytic separation of pre-rRNAs to initiate rRNA production. In yeast, processing of the primary transcript encoding 18 S, 5.8 S, and 25 S rRNAs has been studied extensively. Nevertheless, most nucleases remain to be identified. Here, we show that Rcl1, conserved in all eukaryotes, cleaves pre-rRNA at socalled site A 2, a co-transcriptional cleavage step that separates rRNAs destined for the small and large subunit. Recombinant Rcl1 cleaves pre-rRNA mimics at site A 2 in a reaction that is sensitive to nearby RNA mutations that inhibit cleavage in vivo. Furthermore, mutations in Rcl1 disrupt rRNA processing at site A 2 in vivo and in vitro. Together, these results demonstrate that the role of Rcl1 in eukaryotic pre-rRNA processing is identical to that of RNase III in bacteria: to co-transcriptionally separate the prerRNAs destined for the small and large subunit. Furthermore, because Rcl1 has no homology to other known endonucleases, these data also establish a novel class of nucleases. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.