Fig. 2

The mRNA cleavage and polyadenylation complex is required for snoRNA synthesis. a Schematic of the RNase H cleavage assay used in b–e. After RNase H cleavage of the snoRNA at sites of RNA:DNA hybrids in the presence of a sequence-specific DNA oligonucleotide, the 3′ fragment (mature or 3′-extended) is detected by Northern blotting (NB). Addition of oligo d(T) to the RNase H reaction removes heterogenous poly(A) tails, generating discrete products. b, c Total RNA prepared from the indicated strains that were previously grown in thiamine-supplemented medium for 15 h to deplete Pcf11, Seb1, and Dhp1 was treated with RNase H in the presence of DNA oligonucleotides complementary to snR3 (b) and snR99 (c). RNase H reactions were performed in the presence (+) or absence (−) of oligo d(T). The top panel represents a longer exposure of the middle panel to see 3′-extended (3′-ext) cleavage products. The 5S rRNA was used as a loading control. d, e As described in b, c, but using cells that were previously treated with rapamycin to deplete Ysh1 and Rna14 from the nucleus. f Northern blot analysis using total RNA prepared from the indicated strains that were treated with either rapamycin (lanes 1–3) or thiamine (lanes 4–8). The blot was hybridized using DNA probes specific to snR3 and the 18S rRNA. The position of mature snR3, 18S and 25S rRNAs, as well as snR3 read-through (RT) products is indicated on the right