Release of U18 snoRNA from its host intron requires interaction of Nop1p with the Rnt1p endonuclease

An external stem, essential for the release of small nucleolar RNAs (snoRNAs) from their pre-mRNAs, flanks the majority of yeast intron-encoded snoRNAs. Even if this stem is not a canonical Rnt1p substrate, several experiments have indicated that the Rnt1p endonuclease is required for snoRNA processing. To identify the factors necessary for processing of intron-encoded snoRNAs, we have raised in vitro extracts able to reproduce such activity. We found that snoRNP factors are associated with the snoRNA-coding region throughout all the processing steps, and that mutants unable to assemble snoRNPs have a processing-deficient phenotype. Specific depletion of Nop1p completely prevents U18 snoRNA synthesis but does not affect processing of a dicistronic snoRNA-coding unit that has a canonical Rnt1p site. Correct cleavage of intron-encoded U18 and snR38 snoRNAs can be reproduced in vitro by incubating together purified Nop1p and Rnt1p. Pull-down experiments showed that the two proteins interact physically. These data indicate that cleavage of U18, snR38 and possibly other intron-encoded snoRNAs is a regulated process, since the stem is cleaved by the Rnt1p endonuclease only when snoRNP assembly has occurred.