U3 small nucleolar RNA is essential for cleavage at sites 1, 2 and 3 in pre-rRNA and determines which rRNA processing pathway is taken in Xenopus oocytes

A molecular dissection of U3 small nucleolar RNA (snoRNA) was performed in vivo in Xenopus oocytes and the effects on rRNA processing were analyzed. O ocyte injection of antisense oligonucleotides against parts of U3 snoRNA re sulted in specific fragmentation of U3 by endogenous RNase H. Fragmentation of U3 domain II correlated with a decrease in 20 S pre-rRNA and a concomit ant increase in 36 S pre-rRNA, indicating reduced cleavage at site 3. Conve rsely, fragmentation of U3 domain I completely blocked 18 S rRNA formation, increased the 20 S rRNA precursor, and decreased 36 S pre-rRNA, indicating inhibition of cleavage at sites 1 + 2. rRNA processing defects at sites 1 + 2 or 3 after destruction of intact endogenous U3 snoRNA were rescued by i njection of in vitro transcripts of U3 snoRNA or certain U3 fragments. Thus , cleavage at sites 1 + 2 and 3 is U3 snoRNA dependent. Moreover, U3 snoRNA has two functional modules: domain I for sites 1 + 2 cleavage and domain I I for site 3 cleavage. The data suggest that whichever of these U3 domains acts first determines which rRNA processing pathway will be taken: cleavage first at site 3 of pre-rRNA leads to pathway A, whereas cleavage first at sites 1 + 2 leads to pathway B for rRNA processing. Predictions of this mod el were validated by rescue of site 3 cleavage by injection of just domain II after U3 depletion. Rescue of sites 1 + 2 cleavage required covalent con tinuity of domain I with the hinge region and non-covalent association with domain II. We could experimentally shift which rRNA processing pathway was taken by injecting fragments of U3 to compete with endogenous U3 snoRNA. ( C) 1999 Academic Press.