Loss of RISC proteins leads to accumulation of different pre-5.8S rRNA species. (A) Depiction of probe positions around the 5.8S rRNA region. The probe names are shown under the pre-rRNA. (B) Total RNA was prepared from test cells 48 h after ASO treatment, and subjected to northern hybridization. The same membrane was hybridized sequentially using probes specific to 5′ (left panel, 5′ probe), 5.8S (middle panel, 5.8S probe) and 3′ regions (right panel, 3′ probe), respectively. U3 snoRNA was used as a loading control. Different pre-5.8S rRNA species are indicated. The asterisks indicate RNA species that were only detected with one probe. M, size maker in nucleotides (low range ssRNA ladder, Biolabs); 5.8S-L, the longer version of mature 5.8S rRNA; shorter exposure for 5.8S-L rRNA is shown in left panel, lower part. (C) Summary of changes in the levels of different pre-5.8S rRNA species in cells depleted of the RISC pathway proteins. App. size, estimated nucleotide length of the RNA. Extension, types of 5′ or 3′ flanking sequences in pre-5.8S rRNAs. The larger arrows indicate stronger accumulation.