THE 5' END OF YEAST 5.8S RIBOSOMAL-RNA IS GENERATED BY EXONUCLEASES FROM AN UPSTREAM CLEAVAGE SITE

We have developed techniques for the detailed analysis of cis-acting s equences in the pre-rRNA of Saccharomyces cerevisiae and used these to study the processing of internal transcribed spacer 1 (ITS1) leading to the synthesis of 5.8S rRNA. As is the case for many eukaryotes, the 5' end of yeast 5.8S rRNA is heterogeneous; we designate the major, s hort form 5.8S(S), and the minor form (which is seven or eight nucleot ides longer) 5.8S(L). These RNAs do not have a precursor/product relat ionship, but result from the use of alternative processing pathways. I n the major pathway, a previously unidentified processing site in ITS1 , designated A3, is cleaved. A 10 nucleotide deletion at site A3 stron gly inhibits processing of A3 and the synthesis of 5.8S(S); processing is predominantly transferred to the alternative 5.8S(L) pathway. Site A3 lies 76 nucleotides 5' to the end of 5.8S(S), and acts as an entry site for 5'--> 3' exonuclease digestion which generates the 5' end of 5.8S(S). This pathway is inhibited in strains mutant for XRN1p and RA T1p. Both of these proteins have been reported to have 5'--> 3' exonuc lease activity in vitro. Formation of 5.8S(L) is increased by mutation s at A3 in cis or in RAT1p and XRN1p in trans, and is kinetically fast er than 5.8S(S) synthesis.