FIG. 2.

Naf1p depletion affects 18S rRNA accumulation. GAL::zz-naf1 cells were grown in a medium containing galactose (gal), raffinose, and sucrose, were washed, and were shifted to a medium containing glucose (glu). Cell aliquots were collected from the culture grown in galactose, raffinose, and sucrose (lanes 1) and after 6 (lanes 2), 12 (lanes 3), 24 (lanes 4), 48 (lanes 5) and 72 (lanes 6) h of growth in glucose-containing medium. Total proteins and RNAs were extracted from these samples for Western (A) and Northern (B) blot analysis. (A) Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a cellulose membrane. ZZ-Naf1p was detected by use of rabbit PAP and Nsr1p by using a monoclonal antibody. The third panel corresponds to an overexposed film to show the residual production of ZZ-Naf1p even after prolonged growth on glucose-containing medium. (B) High-molecular-weight RNAs were separated on a formaldehyde-agarose gel, and low-molecular-weight RNAs were separated on a denaturing 6% polyacrylamide gel. Gels were transferred to nylon membranes. Mature rRNAs and pre-rRNA processing intermediates were detected by use of specific oligonucleotide probes. (C) Cartoon of the pre-rRNA processing pathway. In wild-type cells, the 35S pre-rRNA is first cleaved at site A0 within the 5′ external transcribed spacer (5′ ETS), producing intermediate 33S, which is very rapidly cleaved at site A1, the 5′ end of 18S rRNA, to produce intermediate 32S. 32S is then cleaved at site A2 within the internal transcribed spacer 1 (ITS1), releasing 20S, the immediate precursor to 18S rRNA, and 27SA2. 27SA2 is then processed via two alternative pathways. It is either cut at site A3 to produce 27SA3, which is then trimmed by 5′-to-3′ exonucleases up to site B1(S), producing 27SB(S). Alternatively, it can be processed into 27SB(L) by an as-yet-unknown mechanism. 27SB(S) and 27SB(L) are then processed in the same manner to produce 25S and 5.8S(S) or 5.8S(L), respectively. In cells depleted of Naf1p, a fraction of 35S is directly cut at site A3, producing 23S that is degraded by the exosome. For a detailed review of the pre-rRNA processing pathway in yeast, see reference 96.