Slx9p facilitates efficient ITS1 processing of pre-rRNA in Saccharomyces cerevisiae

Abstract

Slx9p (Ygr081cp) is a nonessential yeast protein previously linked genetically with the DNA helicase Sgs1p. Here we report that Slx9p is involved in ribosome biogenesis in the yeast Saccharomyces cerevisiae. Deletion of SLX9 results in a mild growth defect and a reduction in the level of 18S rRNA. Co-immunoprecipitation experiments showed that Slx9p is associated with 35S, 23S, and 20S pre-rRNA, as well as U3 snoRNA and, thus, is a bona fide component of pre-ribosomes. The most striking effects on pre-rRNA processing resulting from deletion of SLX9 is the accumulation of the mutually exclusive 21S and 27SA2 pre-rRNA. Furthermore, deletion of SLX9 is synthetically lethal with mutations in Rrp5p that block cleavage at either site A2 or A3. We conclude that Slx9p has a unique role in the processing events responsible for separating the 66S and 43S pre-ribosomal particles. Interestingly, homologs of Slx9p were found only in other yeast species, indicating that the protein has been considerably less well conserved during evolution than the majority of trans-acting processing factors.

FIGURE 1.

Processing of pre-rRNA in S. cerevisiae. (A) Structure of the 35S pre-rRNA. The mature sequences 18S, 5.8S, and 25S rRNA (thick bars) are separated and flanked by transcribed spacers (thin bars). The positions of the processing sites and the probes used in this study are indicated. (B) The-rRNA processing pathway. The sedimentation coefficients of the pre-ribosomal complexes are indicated at the left and right. Note that the order of the cleavages at the A sites is flexible to some extent (see text). The precise role of the cleavage at site A4 remains to be established.

FIGURE 2.

Amino acid alignment of Slx9p with yeast homologs. Sequence alignments were carried out using CLUSTAL W and BOXSHADE at www.ch.EMBnet.org. Accession numbers for the sequences are as follows: Sc S. cerevisiae Slx9p (UniProt P53251), Sp S. pombe (Q9C0Z3), Cg C. glabrata (Q6FNC2), Kl K. lactis (Q6CT43), Ag A. gossypii (Q758S2), Dh D. hansenii (Q6BMZ5). Identical amino acids are shaded in black, while similar amino acids are in gray.

FIGURE 3.

Identification of RNA species co-immunoprecipitated by ProtA–Slx9p. Total cell extracts from exponentially growing strains slx9::KAN (Y14711), slx9::KAN+pURA3–ProtA::slx9, and SC1413 (TAP::Rio2) were prepared and treated with IgG beads. (A,B) Northern analysis of co-precipitated RNA (IP) or nonbound RNA (SUP) on agarose gels using probe 1 and 2 (see Fig. 1A). (C) Northern analysis of co-precipitated RNA separated on a 8% polyacrylamide gel using a probe complementary to the C′ box of U3 snoRNA.

FIGURE 4.

Northern analysis of pre-rRNA in Δslx9 cells. Total RNA was isolated from exponentially growing YJV140 (wild-type), strain slx9::LEU2 (YCV711) and slx9::LEU2 carrying a plasmid-borne SLX9 gene. The RNA was separated on denaturing 1.2% agarose gels (A,C–E) or on 8% polyacrylamide gels (B). See Figure 1A for the position of the different probes. (A) Northern analysis to visualize 25S and 18S rRNA. The 18S: 25S ratio was determined by Northern analysis using probes that detect 18S and 25S rRNA. The signals obtained by phosphor imaging were quantitated using the ImageQuant software tools. (B) Northern analysis of 5.8S rRNA using probe 4. (C–F) Northern analysis with probes 1–3 and 5 (see Fig. 1A). The same blot was used for the different hybridizations. The asterisk indicates a nonspecific hybridization signal.

FIGURE 5.

Reverse transcription analysis of pre-rRNA processing intermediates. RNA was isolated from strains Y10000 (EUROSCARF wild-type, lane 1), slx9::KAN (Y14711) (lane 2), slx9::LEU2 (YCV711) (lane 3), and slx9::LEU2+pHIS–SLX9 (lane 4) and used as a template for reverse transcription using probe 3 (see Fig. 1A). The bands indicated by the asterisks represent artificial stops.

FIGURE 6.

Effect of the deletion of SLX9 in cells expressing the mutant genes rrp5-Δ3 and rrp5-Δ6. (A) Growth. Cells of strain YJV306 (rrp5-Δ3), YJV207 (rrp5-Δ6), YJV163 (wild-type RRP5), rrp5-Δ3Δslx9 (YRB154Δ3), rrp5-Δ6Δslx9 (YRB154Δ6), and RRP5Δslx9 (YRB154), were streaked out on YPGAL and YPD plates and grown at 30°C for 3–4 d. (B–D) Effect on pre-rRNA processing. The strains were grown on YPGAL to mid-exponential phase and then shifted to YPD medium for 24 h. Total RNA was isolated from equal amounts of cells, separated on 1.2% agarose gels, and subjected to ethidium bromide staining and Northern analysis. (B) Northern analysis to visualize mature 18S and 25S rRNA. The 18S:25S ratio was established by Northern analysis using probes that detect 18S and 25S rRNA. The signals obtained by phosphor imaging were quantitated using the ImageQuant software tools. (C) Northern analysis using probe 1. (D) Northern analysis using probe 2. See Figure 1A for the positions of the probes.