To establish the nature of the observed RNA fragmentation, 3 0 -ends of the products generated by the H 2 O 2mediated cleavage in the 25S at positions +610-611 and +398-403 were determined by the 3 0 RACE. To this end, DNA 'anchor' oligonucleotide (W242) was ligated with T4 RNA ligase to total RNA from untreated and treated W303 cells to prepare cDNA using a primer specific for the anchor (W243). This served as a template to amplify products containing required fragments using the same 3 0 primer and a 5 0 primer that covers the 5 0 -end of 25S RNA starting at position +50 (W241). The ensuing PCR fragments ( Figure 2D ) were cloned into pGEM-Teasy and sequenced. The results of 10 sequenced clones for the cleavage at +610-611 and 19 clones for the cleavage +398-403 are shown in Figure 2E . In the case of the major site (cuts at positions +610-611), this analysis confirms that the 5 0 and 3 0 ends of this degradation product overlap ( Figure 2E , lower panel), which is consistent with the endonucleolytic mechanism of the cleavage. Mapping the 5 0 and 3 0 ends at site +398-403 by primer extension and 3 0 RACE produced a different pattern: these ends do not match ideally but the products with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an 'anchor' oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3 0 primer and the 5 0 primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398-404 (lower) and +610-611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3 0 RACE analysis for fragments cleaved at site +398-403 (19 independent clones) and site +610-611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. (F) Mapping 3 0 ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7D cells treated with 1mM H 2 O 2 (lanes, 2-4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3 0 ends of fragments cleaved at +398-403 (F-I) and at +610-611 (F-II), respectively. Arrows show more defined 3 0 ends of products cleaved at +610-611 for all strains and at +398-403 in the mutants; vertical bar in F-I indicates heterogenous 3 0 ends of products cleaved at +398-403 in wild-type cells. get progressively shorter pointing at the action of 3 0 !5 0 exonucleases ( Figure 2E, upper panel) . The most likely candidate is the exosome, a large complex with a 3 0 !5 0 exonucleolytic activity involved in the processing and degradation of mRNA, rRNA and other RNA substrates (43) . Mutants in the exosome core component Rrp41, the nuclear subunit Rrp6 and the cytoplasmic cofactor Ski7, were used to assess the status of the product 3 0 ends by a specific RNase H cleavage. This cleavage, directed by a DNA-RNA hybrid between oligonucleotide W244 and a complementary region in the 25S starting at residue +271, allows higher resolution of analysed RNAs. This analysis shows that products generated at site +398-403 in the exosome mutants rrp41-1 and ski7D, but not in rrp6D, are 3 0 extended and less heterogenous than corresponding fragments in the wild-type strain (Figure 2F-I; data not shown). In contrast, positions of cleavages at site +610-611 are not affected by mutations in the exosome (Fig. 2F-II) . This suggests that the cytoplasmic exosome may contribute to rRNA decay by digesting 3 0 ends of at least some cleavage products.