FIG. 2.

Tgs1p is required for pre-rRNA processing. (A to C) Northern blot (A and B) and primer extension (C) analyses of pre-rRNA processing defects in tgs1Δ and isogenic wild-type backgrounds. Temperatures of growth (16, 23, and 30°C) and the oligonucleotides used (001 to 006 [Fig. 1A]) are indicated. The ratio of 18S to 25S was quantitated with a PhosphorImager (Typhoon; Amersham Biosciences) and estimated to be ∼2.6 for the wild type and ∼2.1 to ∼2.2 for the mutant. tgs1Δ cells grown under nonpermissive conditions accumulated the D-A₂ spacer fragment that is readily detected in xrn1Δ strains (27) (B). The use of the two alternative pathways of 5.8S-25S synthesis (30) was not affected in tgs1Δ strains, as indicated by the steady-state levels of the two forms of 5.8S rRNA (A) and the conserved ratio of cleavage at site B_1S and B_1L (C). In wild-type cells, the 18S rRNA is dimethylated at its 3′ end on two adjacent adenosines by Dim1p (9). cDNA strands that extended across this position from oligonucleotide 002 revealed that pre-rRNAs accumulated in tgs1Δ cells are dimethylated (C). (D) Steady-state levels of the box C+D snoRNAs U3 and U14 and the box H+ACA snoRNAs snR10 and snR30 in tgs1Δ cells and a wild-type (WT) isogenic control. Total RNA was extracted at 23 and 30°C. Oligonucleotides specific to the snoRNA species targeted were used to probe a Northern blot membrane (see Materials and Methods).