The roles of endonucleolytic cleavage and exonucleolyticdigestion in the 5ⴕ-end processing of S. cerevisiae boxC/D snoRNAsCHRISSIE YOUNG LEE, ALBERT LEE, and GUILLAUME CHANFREAUDepartment of Chemistry and Biochemistry and the Molecular Biology Institute, University of California Los Angeles, LosAngeles, California 90095-1569, USAABSTRACTSmall nucleolar RNAs (snoRNAs) play important roles in ribosomal RNA metabolism. In Saccharomyces cerevisiae, box C/DsnoRNAs are synthesized from excised introns, polycistronic precursors, or independent transcription units. Previous studieshave shown that only a few independently transcribed box C/D snoRNAs are processed at their 5ⴕ end. Here we describe 12additional independently transcribed box C/D snoRNAs that undergo 5ⴕ-end processing. 5ⴕ Extensions found in the precursorsof these snoRNAs contain cleavage sites for Rnt1p, the S. cerevisiae homolog of RNase III, and unprocessed precursorsaccumulate in vivo in the absence of Rnt1p. Rnt1p cleavage products were identified in vivo when the 5ⴕ → 3ⴕ exonucleasesXrn1p and Rat1p are inactivated (xrn1⌬ rat1-1) and in vitro using model RNA substrates and recombinant Rnt1p. Some of thesesnoRNAs show increased levels of unprocessed precursors when the rnt1⌬ deletion is combined to the xrn1⌬ rat1-1 mutation,suggesting that these exonucleases participate in the 5ⴕ processing or the degradation of the snoRNA precursors. Unprocessedprecursors are not significantly destabilized in the absence of the trimethylguanosine capping enzyme Tgs1p, suggesting that a5ⴕ monomethyl cap is sufficient to ensure stabilization of these precursors. These results demonstrate that the majority ofindependently transcribed box C/D snoRNAs from the yeast genome undergo 5ⴕ-end processing and that the Rnt1p endonucle-ase and the Xrn1p and Rat1p 5ⴕ → 3ⴕexonucleases have partially redundant functions in the 5ⴕ-end processing of these snoRNAs.Keywords: Endoribonucleases; exoribonuclease; Rnt1p; RNase III; stem–loopINTRODUCTIONSmall nucleolar RNAs (snoRNAs) are essential cofactors inribosomal RNA (rRNA) metabolism. A few snoRNAs arenecessary for cleavage steps in the maturation of the 35SrRNA precursor, but most of them are required to guidebases or sugar modifications within the rRNA precursor(Tollervey and Kiss 1997; Kiss 2001). Small nucleolar RNAsare subdivided in two major structural families (Balakin etal. 1996; Ganot et al. 1997b). Box C/D snoRNAs guide themethylation of the ribose 2⬘ hydroxyl groups of nucleotidesin the 35S rRNA precursor (Cavaille et al. 1996; Kiss-Laszloet al. 1996). H/ACA snoRNAs guide the conversion of uri-dines to pseudouridines in the rRNA precursor (Ganot et al.1997a; Ni et al. 1997).Small nucleolar RNAs are usually produced by posttran-scriptional processing from precursors species (Tollerveyand Kiss 1997). Although most mammalian snoRNAs areencoded within intron sequences and processed from eitherunspliced precursors or lariat species, only a few yeastsnoRNAs are intron encoded (U18, U24, snR34, snR38,snR39, snR44, snR54, and snR59). Most yeast small RNAsare either generated from independent transcription unitsor initially transcribed as polycistronic units. In the case ofpolycistronic transcription units, processing intermediatesare generated by RNase III cleavage in the spacer separatingeach snoRNA from the other (Chanfreau et al. 1998a,b; Quet al. 1999). The resulting intermediates carrying only onesnoRNA sequence are further processed by exonucleases(Petfalski et al. 1998; Qu et al. 1999) to generate the matureends.The initial transcripts of independently transcribed boxC/D snoRNAs all possess a trimethylguanosine (TMG) cap.The snoRNAs that are not processed at their 5⬘ end such asU3, snR4, and snR13 will retain the TMG cap structure(Samarsky and Fournier 1999). Those that undergo 5⬘-endReprint requests to: Guillaume Chanfreau, Department of Chemistryand Biochemistry and the Molecular Biology Institute, University of Cali-fornia Los Angeles, Box 951569, Los Angeles, CA 90095-1569, USA; e-mail:[email protected]; fax: (310) 206-4038.Article and publication are at http://www.rnajournal.org/cgi/doi/10.1261/rna.5126203.RNA (2003), 9:1362–1370. Published by Cold Spring Harbor Laboratory Press. Copyright © 2003 RNA Society.1362processing, such as snR39b, snR40, snR47, and snR79 (Z9),will lose the TMG cap structure (Chanfreau et al. 1998a). Inthese documented cases, the yeast ortholog of RNase III,Rnt1p, cleaves the 5⬘ extension found in the precursor ofthese snoRNAs, and provides an entry site for exonucleo-lytic digestion. The exonucleases responsible for the finaltrimming step have not been identified, but have beenspeculated to be Xrn1p and/or Rat1p (Chanfreau et al.1998a).Because of the limited number of independently tran-scribed box C/D snoRNA genes described, it is not clearwhether 5⬘-end processing is a common pathway or limited to a few snoRNAs. In this study, we examine the question ofthe processing of recently identified independently tran-scribed box C/D snoRNAs. We show that most of themundergo 5⬘-end processing by a combination of endonu-cleolytic cleavage by Rnt1p and exonucleolytic digestion byXrn1p and/or Rat1p. These results show that the majority ofindependently transcribed box C/D snoRNAs undergo 5⬘-end processing, and that the enzymes involved in their pro-cessing have partially redundant functions.RESULTSIn silico detection of putative Rnt1p target sitesupstream from recently identified box C/DsnoRNA genesWe analyzed the predicted secondary structure of sequencesfound upstream of box C/D snoRNA gene sequences iden-tified through computational screening (Lowe and Eddy1999; Samarsky and Fournier 1999). This analysis revealed the presence of putative stem–loop structures upstreamfrom the independently transcribed box C/D snoRNAssnR50, snR52, snR58, snR60, snR62, snR63, snR64, snR65,snR66, snR68, snR69, and snR71 (Fig. 1). Most of thesestem–loop structures are capped by tetraloops showing theconsensus sequence AGNN. One exception was snR71,which showed a GGUU tetraloop se-quence. In three cases (snR50, snR60,and snR69), the predicted secondarystructures were formed by coaxial stack-ing of a short stem–loop carrying theAGNN tetraloop onto a longer stem(Fig. 1). The Saccharomyces cerevisiaehomolog of RNase III, Rnt1p, specifi-cally cleaves double-stranded structurescapped by tetraloop with the sequenceAGNN, and the enzyme