The roles of endonucleolytic cleavage and exonucleolytic digestion in the 5′-end processing of S. cerevisiae box C/D snoRNAs

Abstract

Small nucleolar RNAs (snoRNAs) play important roles in ribosomal RNA metabolism. In Saccharomyces cerevisiae, box C/D snoRNAs are synthesized from excised introns, polycistronic precursors, or independent transcription units. Previous studies have shown that only a few independently transcribed box C/D snoRNAs are processed at their 5′ end. Here we describe 12 additional independently transcribed box C/D snoRNAs that undergo 5′-end processing. 5′ Extensions found in the precursors of these snoRNAs contain cleavage sites for Rnt1p, the S. cerevisiae homolog of RNase III, and unprocessed precursors accumulate in vivo in the absence of Rnt1p. Rnt1p cleavage products were identified in vivo when the 5′ → 3′ exonucleases Xrn1p and Rat1p are inactivated (xrn1Δ rat1-1) and in vitro using model RNA substrates and recombinant Rnt1p. Some of these snoRNAs show increased levels of unprocessed precursors when the rnt1Δ deletion is combined to the xrn1Δ rat1-1 mutation, suggesting that these exonucleases participate in the 5′ processing or the degradation of the snoRNA precursors. Unprocessed precursors are not significantly destabilized in the absence of the trimethylguanosine capping enzyme Tgs1p, suggesting that a 5′ monomethyl cap is sufficient to ensure stabilization of these precursors. These results demonstrate that the majority of independently transcribed box C/D snoRNAs from the yeast genome undergo 5′-end processing and that the Rnt1p endonuclease and the Xrn1p and Rat1p 5′ → 3′exonucleases have partially redundant functions in the 5′-end processing of these snoRNAs.

Keywords

• Endoribonucleases
• exoribonuclease
• Rnt1p
• RNase III
• stem–loop