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 (snoRNA s) from their pre-mRNAs, flanks the majority of yeast intron-encoded snoRNA s. Even if this stem is not a canonical Rnt1p substrate, several experiment s have indicated that the Rnt1p endonuclease is required for snoRNA process ing. To identify the factors necessary for processing of intron-encoded sno RNAs, we have raised in vitro extracts able to reproduce such activity. We found that snoRNP factors are associated with the snoRNA-coding region thro ughout all the processing steps, and that mutants unable to assemble snoRNP s have a processing-deficient phenotype. Specific depletion of Nop1p comple tely prevents U18 snoRNA synthesis but does not affect processing of a dici stronic snoRNA-coding unit that has a canonical Rnt1p site. Correct cleavag e of intron-encoded U18 and snR38 snoRNAs can be reproduced in vitro by inc ubating together purified Nop1p and Rnt1p. Pull-down experiments showed tha t the two proteins interact physically. These data indicate that cleavage o f U18, snR38 and possibly other intron-encoded snoRNAs is a regulated proce ss, since the stem is cleaved by the Rnt1p endonuclease only when snoRNP as sembly has occurred.