2 DISTINCT RECOGNITION SIGNALS DEFINE THE SITE OF ENDONUCLEOLYTIC CLEAVAGE AT THE 5'-END OF YEAST 18S RIBOSOMAL-RNA

Three of the four eukaryotic ribosomal RNA molecules (18S, 5.8S and 25 -28S rRNA) are transcribed as a single precursor, which is subsequentl y processed into the mature species by a complex series of cleavage an d modification reactions, Early cleavage at site A1 generates the matu re 5'-end of 18S rRNA, Mutational analyses have identified a number of upstream regions in the 5' external transcribed spacer (5' ETS), incl uding a U3 binding site, which are required in cis for processing at A 1. Nothing is known, however, about the requirement for cis-acting ele ments which define the position of the 5'-end of the 18S rRNA or of an y other eukaryotic rRNA. We have introduced mutations around A1 and an alysed them in vivo in a genetic background where the mutant pre-rRNA is the only species synthesized. The results indicate that the mature 5'-end of 18S rRNA in yeast is identified by two partially independent recognition systems, both defining the same cleavage site. One mechan ism identifies the site of cleavage at A1 in a sequence-specific manne r involving recognition of phylogenetically conserved nucleotides imme diately upstream of A1 in the 5' ETS. The second mechanism specifies t he 5'-end of 18S rRNA by spacing the A1 cleavage at a fixed distance o f 3 nt from the 5' stem-loop/pseudoknot structure located within the m ature sequence. The 5' product of the A1 processing reaction can also be identified, showing that, in contrast to yeast 5.8S rRNA, the 5'-en d of 18S rRNA is generated by endonucleolytic cleavage.