Xenopus U3 snoRNA GAC-box A′ and box A sequences play distinct functional roles in rRNA processing

Mutations in the 5 ' portion of Xenopus U3 snoRNA were tested for function in oocytes. The results revealed a new cleavage site (AO) in the 3 ' region of vertebrate external transcribed spacer sequences. In addition, U3 mutagenesis uncoupled cleavage at sites 1 and 2, flanking the 5 ' and 3 ' ends of 18S rRNA, and generated novel intermediates: 19S and 18.5S pre-rRNAs. Furthermore, specific nucleotides in Xenopus U3 snoRNA that are required for cleavages in pre-rRNA were identified: box A is essential for site AO cleavage, the GAC-box A ' region is necessary for site I cleavage, and the 3 ' end of box A ' and flanking nucleotides are required for site 2 cleavage. Differences between metazoan and yeast U3 snoRNA-mediated rRNA processing are enumerated. The data support a model where metazoan U3 snoRNA acts as a bridge to draw together the 5 ' and 3 ' ends of the 18S rRNA coding region within pre-rRNA to coordinate their cleavage.