Processing of the Intron-Encoded U18 Small Nucleolar RNA in the Yeast Saccharomyces cerevisiaeRelies on Both Exo- and Endonucleolytic Activities

ABSTRACT

Many small nucleolar RNAs (snoRNAs) are encoded within introns of protein-encoding genes and are released by processing of their host pre-mRNA. We have investigated the mechanism of processing of the yeast U18 snoRNA, which is found in the intron of the gene coding for translational elongation factor EF-1β. We have focused our analysis on the relationship between splicing of the EF-1β pre-mRNA and production of the mature snoRNA. Mutations inhibiting splicing of the EF-1β pre-mRNA have been shown to produce normal U18 snoRNA levels together with the accumulation of intermediates deriving from the pre-mRNA, thus indicating that the precursor is an efficient processing substrate. Inhibition of 5′→3′ exonucleases obtained by insertion of G cassettes or by the use of a rat1-1 xrn1Δ mutant strain does not impair U18 release. In the Exo⁻ strain, 3′ cutoff products, diagnostic of an endonuclease-mediated processing pathway, were detected. Our data indicate that biosynthesis of the yeast U18 snoRNA relies on two different pathways, depending on both exonucleolytic and endonucleolytic activities: a major processing pathway based on conversion of the debranched intron and a minor one acting by endonucleolytic cleavage of the pre-mRNA.

ACKNOWLEDGMENTS

We thank Josette Banroques for hospitality in her laboratory to T.V. in the initial part of this work and Stephen Kearsey and David Tollervey for providing the Exo⁻ strain. We also thank Alessandro Michienzi for helpful discussions, Massimo Arceci for his skillful technical help, and Fabio Riccobono and M-Medical for oligonucleotide facilities.

This work was partially supported by grants from Biotecnologie MURST 5%, C.N.R. Target Project on Biotechnology, and PRIN 40%-MURST.