Strains spb1-1 and spb1-2 are complemented by the wild-type SPB1 gene, and both behave as suppressors of pab1Δ. (A) The wild-type SPB1 gene can restore normal growth of both spb1-1 and spb1-2. Strains spb1-1 (YAS 151), spb1-2 (JR4710), or these strains transformed by the plasmid pAS16 that contains the wild-type SPB1 gene (YBL4383 and YBL4384, respectively) were streaked onto yeast-peptone-dextrose (YPD) plates and grown at 17°C (for 7 days) or at 37°C (for 3 days). (B) Double mutants spb1-1 pab1Δ (YBL4459) and spb1-2 pab1Δ (YBL4258) were streaked onto a YPD plate, along with a single pab1Δ strain as a control. These three strains harbor the plasmid pBL471 that bears a wild-type PAB1 gene that complements the absence of PAB1 on their chromosome and a URA3 gene as a marker. When streaked onto 5-FOA-containing plates, the pab1Δ strain is unable to grow due to its inability to lose the PAB1 gene (plasmid pBL471 containing the URA3 gene), while both spb1-1 and spb1-2 are able to lose this plasmid and thus to grow on these plates.

Characterization of the SPB1 gene. (A) Total RNA from a wild-type W303 strain was probed in a Northern blot experiment with a fragment of the SPB1 gene. The positions of the 25S and 18S rRNA are indicated on the right of the lane. A single band, one corresponding to an mRNA of 2.9 kb, was detected by this probe. (B) One of the two copies of the SPB1 gene was deleted in a diploid strain by insertion of a TRP1 marker (YBL4460). These cells were then sporulated and the four daughter spores were dissected by micromanipulation and grown on YPD plates. In each tetrad, only two spores were able to grow, all of which were Trp⁻, indicating that the gene is essential for spore development.

Sequence alignment of the predicted AdoMet-binding region of Spb1p from S. cerevisiae with potential homologues from different species. Eight predicted proteins from different organisms possess significant similarities with the N-terminal moiety of Spb1p from S. cerevisiae (Sc; accession number, CAA42391) as follows: Bc, Botryotinia fuckeliana (AL111693); Sp, Schizosaccharomyces pombe pmt2 (CAA22605); Mm, Mus musculus (AI574417); Cp, Cryptosporidium parvum (AQ450296); At, Arabidopsis thaliana (CAB39594); Hs, Homo sapiens JM23 (HSA005892); Ce, Caenorhabditis elegans (CAA85279); and Ec, E. coli (74) (AAC76211). Residues identical between the different species are on a black background, and the ones that are similar are on a gray background. The four motifs (I, post I, II, and III) that form the putative AdoMet-binding domain are boxed.

Polysome profile analysis. Cellular extracts were loaded onto 15 to 50% sucrose gradients and centrifuged for 2 h at 275,0000 × g. A continuous A₂₆₀ record for each gradient is presented with the top of the tube on the left. Extracts were prepared from the following: wt, wild-type strain (YBL4164); spb1-2 25°C, the spb1-2 strain (JR4710) grown at the permissive temperature of 25°C; and spb1-2 37°C, the spb1-2 strain grown for 1 h at the nonpermissive temperature of 37°C. The arrows indicate the peaks of 40S, 60S, and 80S subunits and of the half-mer.

Scheme of pre-rRNA processing in yeast. (A) Structure of the rRNA transcript with the cleavage sites and the location of the hybridization probes. The primary transcript of 35S contains the 5′ external transcribed spacer, followed by the 18S rRNA, the ITS1, the 5.8S rRNA, the ITS2, the 25S rRNA, and the 3′ external transcribed spacer. The mature species of 18S, 5.8S, and 25S are represented by thick lines, and the spacers are represented by thin lines. The location of the three hybridization primers is also shown (Oligo 1, Oligo 2, and Oligo 3). (B) Simplified pre-rRNA processing scheme. The 35S pre-rRNA is cleaved successively at sites A0 and A1 to yield the 33S and 32S pre-rRNA species. Then, cleavage at A2 frees the 20S form that can further be processed into 18S rRNA and the 27SA2 that undergoes a more complex pathway. Cleavage at A3 leads to 27SA3 that can be processed into two different forms. In this simplified scheme, only 27SB is represented that will lead to 7S and 25S after cleavage at C1 and C2. 7S will be further processed into 5.8S.

Pulse-chase labeling of the pre-rRNA. Cells were grown either at 25°C or heat-shocked for 1 h at 37°C; they were then labeled for 2 min with tritiated methionine and chased with cold methionine for the indicated times. Total RNA was extracted and separated on a denaturing 1.2% agarose gel that was transferred to a nylon membrane and exposed with an intensifying screen for three days. Lanes 1 to 3 and 7 to 9, wild-type strain (YBL4164); lanes 4 to 6 and 10 to 12, spb1-2 (YBL4166); lanes 1 to 6, cells grown at 25°C; lanes 7 to 12, cells grown at 37°C.

Northern blot hybridization of the various pre-rRNA intermediate species. Pre-rRNA intermediates can be detected by Northern blotting by using probes complementary to various regions of the different spacers. Total RNA was extracted from wild-type (YBL4164) or spb1-2 (YBL4166) strains grown either at a permissive temperature (25°C) or heat-shocked for 1 h at a nonpermissive temperature (37°C) and tested by hybridization with oligonucleotide probes 1, 2, and 3 as indicated (see Fig. 5 for the positions of the probes). Equivalent amounts of total RNA extracted from the wild-type and the spb1-2 strains were loaded in each lane and visualized by methylene blue staining (left panel). Positions of the major bands are indicated on the left. Panel E corresponds to an overexposure of panel D in order to clearly detect the 7S species that is no longer accumulated in the spb1-2 strain grown at the nonpermissive temperature.

Indirect immunofluorescence detection of Spb1p within the nucleolus. Cells expressing HA-Spb1p (YDK14-1A/pDK423) were fixed with paraformaldehyde and processed for immunofluorescence as described in Materials and Methods. DAPI was added to the mounting medium to visualize the DNA within the mitochondria and the nucleus (DAPI). Anti-HA rat antibodies detect the HA peptide fused to Spb1p within the nucleolus (secondary anti-rat antibody coupled to Texas red). (A) A large field showing several cells labelled with DAPI; panel (D) the same cells observed through a Texas red filter show that HA-Spb1p is mostly localized to the nucleolus. Triple-labeling experiments have been performed by also using a mouse monoclonal antibody raised against Nop1p as a nucleolar marker. (B) Higher magnification of a cell photographed by using interferential contrast (Nomarski); (C) the same cell stained with DAPI reveals the crescent shape zone that corresponds to the nucleolus; (E) anti-Nop1p decorates the nucleolus (secondary anti-mouse antibody coupled to fluorescein); (F) anti-HA-Spb1p gives a signal that overlaps mostly with the signal obtained with the anti-Nop1p antibodies. Arrows show the positions of the nucleoli.

Spb1p is eluted with Nop1p on a Mono-S column. Cellular extracts containing HA-Spb1p (YDK14-1A/pDK376) were fractionated by size chromatography on a Sephadex column. A high-molecular-weight fraction containing HA-Spb1p was then loaded onto a cation-exchange column (Mono-S). Elution was then performed with a gradient of NaCl that was applied in two steps. Fractions of 1 ml were collected, and a sample of each fraction was subjected to an SDS–10% PAGE, transferred to nitrocellulose, and probed with antibodies revealing HA-Spb1p (upper panel), Nop1p (middle), or the ribosomal protein Qsr1 (lower panel). Load, the fraction that was applied onto the Mono-S column; FT, flowthrough fraction that was not retained by the column; lanes 1 to 15, first slope of the gradient from 0 to 0.5 M NaCl; lanes 16 to 23, second slope of the gradient from 0.5 to 1 M NaCl.

Spb1p is associated with Nop1p and Nop5/58p as shown by coimmunoprecipitation. (A) Cellular extracts prepared from a strain expressing HA-Spb1p (YDK14-1A/pDK376) were incubated with various antibodies, and the complexes were precipitated with protein A-Sepharose beads. After several extensive washes, proteins were eluted from the beads, separated by SDS–10% PAGE, and analyzed by Western blotting with anti-HA antibodies that detect HA-Spb1p. Lane 1, total cell extract corresponding to one-tenth the amount of protein used for the immunoprecipitation; lanes 2 to 5, pellets from the immunoprecipitation. Lane 2, anti-Nop1p; lane 3, anti-HA; lane 4, anti-Nop5/58; lane 5, no antibody added. MW, molecular weight markers in thousands. (B) Cellular extracts prepared from strains expressing either wild-type Spb1p or HA-tagged Spb1p were immunoprecipitated with the anti-HA antibodies. Supernatants (S) and pellets (P) were then analyzed by Western blotting with the anti-HA antibodies (upper panel) or with the anti-Nop1p antibodies (lower panel). Lane 1, total cell extract corresponding to one-tenth the amount of protein used for the immunoprecipitation; lanes 2 and 3, strain YDK14-1A/pDK423 that expresses HA-Spb1p; lanes 4 and 5, strain YDK14-1A plus pDK403 that expresses wild-type Spb1p. Lanes 2 and 4, one-tenth the supernatants; lanes 3 and 5, pellets. MW, molecular weight markers in thousands.

Steady-state level of Nop1p and Qsr1p upon Spb1p depletion. A strain expressing HA-Spb1p under the control of a GAL promoter (YDK14-1A/pDK376) was grown in galactose until early log phase and then transferred to glucose-containing medium to turn off the expression of Spb1p. Samples were taken at different times after the shift, and proteins were extracted. Equal amounts of proteins for each time point were separated by SDS–10% PAGE. Western blotting with anti-HA antibodies detects HA-Spb1p migrating at 120 kDa (upper panel). The anti-Nop1p revealed the protein at 38 kDa (middle panel), and Qsr1p was detected at 20 kDa (lower panel). MW, molecular weight markers in thousands.

Spb1p is radiolabelled after UV cross-linking with [³H]AdoMet. Western blotting with anti-HA antibodies revealed the presence of HA-Spb1p in a total cellular extract prepared from the strain (YDK14-1A/pDK376), along with two degradation products (lane 1). The anti-HA antibodies cross-linked to the protein A-Sepharose beads were used to immunoprecipitate HA-Spb1p from the same extract. The beads were then incubated with [³H]AdoMet before being UV cross-linked for 30 min. Proteins were recovered in a denaturing buffer and analyzed by Western blotting (lane 2). The same experiment was performed with a strain in which Spb1p is not tagged (lane 3). The membrane was exposed for autoradiography for 3 weeks at −70°C with an intensifying screen to reveal the tritiated molecules. Lanes 4 and 5 are identical to lanes 2 and 3. MW, molecular weight markers in thousands.