RECOGNITION OF CLEAVAGE SITE A(2) IN THE YEAST PRE-RIBOSOMAL-RNA

Processing of the yeast pre-rRNA at site A(2) in internal transcribed spacer 1 (ITS1) has been shown to require several small nucleolar ribo nucleoprotein particles (snoRNPs) as trans-acting factors. Here we rep ort a detailed mutational analysis of the cis-acting signals required to specify the site of A(2) cleavage. Initial mutations established th at the signals required for accurate cleavage of site A(2) lie in the 3'-flanking sequence; deletion or substitution of nucleotides in this region strongly inhibits processing, and residual cleavage is inaccura te at the nucleotide level. In contrast, the deletion of the 5'-flanki ng nucleotides has no detectable effect on processing. An evolutionari ly conserved sequence, ACAC, is located at the site of cleavage. Subst itution of the 3' AC leads to heterogeneous cleavage, with activation of cleavage at an upstream ACAC sequence. In all mutants that retain a n ACAC element, a site of cleavage is detected immediately 5' to this sequence, showing that this element is recognized. An ACAC sequence is , however, not essential for accurate cleavage of site A(2). An additi onal signal is also present 3' to A(2), in a region that has the poten tial to form a stem-loop structure that is evolutionarily conserved, b ut of low stability. As has been found for site A(1) (the 5' end of th e yeast 18S rRNA), the identification of the site of processing at A(2 ) relies on multiple recognition elements.