Late cytoplasmic maturation of the small ribosomal subunit requires RIO proteins in Saccharomyces cerevisiae

Abstract

Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation. We have recently demonstrated that Rrp10p/Rio1p is such a factor. By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p. Rio2p homologues were identified throughout the Archaea and metazoan species. We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation. In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature. Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles. These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.

FIG. 1.

Rio2p is a member of RIO family and is conserved from archaebacteria to humans. (A) Sequence comparison of the RIO domains of the S. cerevisiae proteins. The E-values of these domains in comparison with the consensus defined in the SMART database are indicated. Black shading highlights amino acids that are identical or similar in all sequences, and dark grey and light grey shading highlights those that are identical or similar in some sequences, respectively. (B) Phylogenetic tree and domain profile of all RIO family members. The phylogenetic tree was established with the ClustalW program and drawn with the Phylodendron program. The RIO domain (SMART) is represented by an oval, and Pfam-B_3091 domain by a circle, and lysine-rich regions by a squared box. Eukaryotes are noted in bold. Accession numbers of members of the RIO1/RRP10 family are: Rio1p_Sc, CAA65511; Rio1p_En, AAC26079; Rio1p_Sp, CAA15723; Rio1p_Ce1, P34649; Rio1p_Ce2, AAC17564; Rio1p_Dm1, AAF50965; Rio1p_Dm2, AAF50033; Rio1p_Ap, BAA79728; Rio1p_Mj, Q57886; Rio1p_Mt, AAB85501; Rio1p_Pa, CAB49514; Rio1p_Ph, BAA30679; Rio1p_At1, Q9FHT0; Rio1p_At2, Q9SK34; Rio1p_Hs1, O14730; Rio1p_Ss, CAC24438; and Rio1p_Hs2, Q9H2L9. Accession numbers of members of the RIO2 family are: Rio2p_Sc, NP_014192; Rio2p_Hs, FLJ11159; Rio2p_Ce; CAB60851.1; Rio2p_Sp, CAB66449.1; Rio2p_Dm, CG11859; Rio2p_At, T45758; Rio2p_Mm, NP_080210; Rio2p_Ap, E72539; Rio2p_Mj, H64433; Rio2p_Pa, B75068; Rio2p_Ph, C71164; and Rio2p_Aj, NP_071248. Species abbreviations: Sc, Saccharomyces cerevisiae; En, Emericella nidulans; Sp, Schizosaccharomyces pombe; At, Arabidopsis thaliana; Hs, Homo sapiens; Ms, Mus musculus; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Ap, Aeropyrum pernix; Mj, Methanococcus jannaschii; Mt, Methanobacterium thermoautotrophicum; Pa, Pyrococcus abyssi; Ph, Pyrococcus horikoshii; Ss, Sulfolobus solfataricus; Af, Archaeoglobus fulgidus. Bold bars on the right side of the figure, 100 amino acids; the bar at the upper right side measures the divergence index.

FIG. 2.

RIO2 gene expression under the control of the inducible GAL10 promoter. (A) Schematic representation of the GAL::RIO2::PROTA allele. (B) Growth of strain YO471 carrying the GAL::RIO2::PROTA allele at 30°C. Cells from exponentially growing cultures in YPG medium (Gal) were harvested, washed, and resuspended either in YPD medium (Glu, triangles) or in galactose medium (Gal, crosses), and incubation was pursued for up to 24 h at 30°C. Cell density was measured at regular intervals, and the cultures were periodically diluted to keep them in the exponential phase of growth. The generation time in YPG is similar to that of an isogenic RIO2⁺ strain. (C) Depletion of Rio2p during growth in YPD medium. Cell extracts of strain YO471 carrying the GAL::RIO2::PROTA allele were prepared from samples harvested at the indicated time points. Rio2p concentration was determined by Western blotting. Equal amounts of total proteins were loaded in every lane, as estimated by Coomassie staining of the gels.

FIG. 3.

(A) Pre-rRNA processing in S. cerevisiae. In the 35S pre-rRNA, the primary transcript, the sequences of the mature 18S, 5.8S, and 25S rRNAs are flanked by the external transcribed spacers (5′ and 3′ ETS) and separated by the internal transcribed spacers (ITS1 and ITS2). Cleavage sites are indicated by uppercase letters A to E, and oligonucleotide probes used in Northern blot hybridizations are indicated by the numbers 1 to 7. Pre-rRNA processing pathway: sequential cleavages of the 35S pre-rRNA at sites A₀ and A₁ generate the 33S and 32S pre-rRNAs. Cleavage of the 32S pre-rRNA at site A₂ in ITS1 yields the 27SA₂ and 20S pre-rRNAs, which are precursors to the RNA components of the large and small ribosomal subunits, respectively. The 27SA₂ precursor is either processed at site A₃ by RNase MRP (major pathway) and then to site B1s by the 5′-3′ exonucleases Rat1p and Xrn1p, or processed at site B_1L (minor pathway), yielding, respectively, the 27SB_S and 27SB_L pre-rRNAs. These two intermediates follow the same processing pathway to 25S and 5.8S_S/L through cleavage at site C₂ in ITS2, followed by 3′-5′ exonucleolytic digestion of 7S_S and 7S_L from site C₂ to E by the exosome complex, and 5′-3′ exonucleolytic digestion to the 5′ end of the 25S rRNA by the exonuclease Rat1p. The final maturation of the 20S pre-rRNA by an endonucleolytic cleavage at site D, occurs after pre-SSU export to the cytoplasm, and produces the mature 18S rRNA and a fragment D-A₂ (5′ ITS1). The D-A₂ fragment is then degraded by the 5′-3′ exonuclease Xrn1p. (B and C) Depletion of Rio2p specifically affects the steady-state level of mature 18S rRNA and results in 20S pre-rRNA accumulation. YO471 (GAL::RIO2) cells were grown in YPG medium (Gal) or in YPD medium (Glu) for 15 h. Total RNA was extracted and separated in 1% agarose-formaldehyde gels to analyze 35S, 32S, 27SA₂, 25S, 20S, and 18S species. Equal amounts of total RNA (5 μg) were loaded in every lane and analyzed. (B) Ethidium bromide staining of the gel. (C) Northern blot analyses of pre-rRNA processing. After transfer to nylon membranes of electrophoresed material, the membranes were hybridized with different probes, as indicated.

FIG. 3.

(A) Pre-rRNA processing in S. cerevisiae. In the 35S pre-rRNA, the primary transcript, the sequences of the mature 18S, 5.8S, and 25S rRNAs are flanked by the external transcribed spacers (5′ and 3′ ETS) and separated by the internal transcribed spacers (ITS1 and ITS2). Cleavage sites are indicated by uppercase letters A to E, and oligonucleotide probes used in Northern blot hybridizations are indicated by the numbers 1 to 7. Pre-rRNA processing pathway: sequential cleavages of the 35S pre-rRNA at sites A₀ and A₁ generate the 33S and 32S pre-rRNAs. Cleavage of the 32S pre-rRNA at site A₂ in ITS1 yields the 27SA₂ and 20S pre-rRNAs, which are precursors to the RNA components of the large and small ribosomal subunits, respectively. The 27SA₂ precursor is either processed at site A₃ by RNase MRP (major pathway) and then to site B1s by the 5′-3′ exonucleases Rat1p and Xrn1p, or processed at site B_1L (minor pathway), yielding, respectively, the 27SB_S and 27SB_L pre-rRNAs. These two intermediates follow the same processing pathway to 25S and 5.8S_S/L through cleavage at site C₂ in ITS2, followed by 3′-5′ exonucleolytic digestion of 7S_S and 7S_L from site C₂ to E by the exosome complex, and 5′-3′ exonucleolytic digestion to the 5′ end of the 25S rRNA by the exonuclease Rat1p. The final maturation of the 20S pre-rRNA by an endonucleolytic cleavage at site D, occurs after pre-SSU export to the cytoplasm, and produces the mature 18S rRNA and a fragment D-A₂ (5′ ITS1). The D-A₂ fragment is then degraded by the 5′-3′ exonuclease Xrn1p. (B and C) Depletion of Rio2p specifically affects the steady-state level of mature 18S rRNA and results in 20S pre-rRNA accumulation. YO471 (GAL::RIO2) cells were grown in YPG medium (Gal) or in YPD medium (Glu) for 15 h. Total RNA was extracted and separated in 1% agarose-formaldehyde gels to analyze 35S, 32S, 27SA₂, 25S, 20S, and 18S species. Equal amounts of total RNA (5 μg) were loaded in every lane and analyzed. (B) Ethidium bromide staining of the gel. (C) Northern blot analyses of pre-rRNA processing. After transfer to nylon membranes of electrophoresed material, the membranes were hybridized with different probes, as indicated.

FIG. 4.

Depletion of Rio2p-protein A results in reduced synthesis of 18S rRNA. (A) Strain YO511 (RIO2⁺) was grown at 30°C in YNB Glu without uracil. (B) Strain YO471 (GAL::RIO2) was grown at 30°C in YPG medium (Gal), then shifted for 12 h to YPD medium (Glu), and finally grown for 7 h in YNB Glu without uracil. Cells were labeled for 4 min with [5,6-³H]uracil and chased with a large excess of unlabeled uracil for 1 to 20 min.

FIG. 5.

Depletion of Rio2p results in accumulation of 20S pre-rRNA in the cytoplasm. The 5′ part of ITS1 (red) was detected by fluorescence in situ hybridization in GAL::RIO1 cells (YO296) (C and D) and GAL::RIO2 (YO470) cells (E and F). Cells were grown either in galactose medium or for 15 h in the presence of glucose. The nucleoplasm was counterstained with DAPI (blue). In wild-type cells (A), the ITS1 is mainly present in the nucleolus and is faintly detected in the cytoplasm. In contrast to crm1-1 cells (YO419) shifted for 2 h at 37°C (B), which display a strong signal in the nucleus and no labeling of the cytoplasm, cells depleted of Rio1p or Rio2p show a build-up of the amount of 20S pre-rRNA in the cytoplasm (D and F).

FIG. 6.

Sedimentation profile of Rio2p-protein A, localization of GFP-Rio2p, and association of Rio2p with 20S pre-rRNA. (A) Subcellular localization of GFP-tagged proteins: GFP-Rio2p exhibits a cytoplasmic and nuclear pattern of fluorescence. GFP-Rio1p shows a cytoplasmic localization, and for Rrp8p-GFP, a punctate pattern characteristic of nucleolar staining is observed. Positions of nuclei were determined by DAPI staining (blue). Overlay images are shown by superposition of the blue and green stains. (B) Sedimentation profiles of Rio2p-protein A and protein A-Rio1p, small-subunit ribosomal protein rpS2, and large-subunit ribosomal protein rpL3 in glycerol gradients. A total extract was loaded on 10 to 30% glycerol gradients and subjected to centrifugation. Fractions were collected, and proteins in each fraction were precipitated with trichloroacetic acid, separated by SDS-PAGE, and revealed by Western blotting. Fractions containing the peak of 40S and 60S ribosomal subunits and 80S ribosomes are indicated. (C) 20S pre-rRNA selectively copurifies with Rio2-protein A. We probed 1/400 of the clarified cell extract (T) or the bulk of the immunoprecipitated RNA (IP) with a mixture of probes 1 and 6 shown in Fig. 3A (18S and 25S) and a D-A₂ probe prepared by multiprime labeling (20S). The fraction of each rRNA or pre-rRNA recovered in the immunoprecipitate was determined by phosphoimager quantification.

FIG.7.

Localization and sedimentation profiles of GFP-Rio1p and GFP-Rio2p in a crm1-1 genetic context. crm1-1 strains used carry plasmids expressing either GFP-Rio2p (A), GFP-Rio1p (B), or GFP (C). Exponentially growing cultures in YNB medium at 25°C were divided in two parts; one part was shifted to 37°C for 2 h, while the other was kept at 25°C, and the subcellular localization of the tagged proteins was analyzed. Subcellular localization of GFP-Rio2p (A) and GFP-Rio1p (B) exhibited a wild-type localization pattern in crm1-1 cells grown at 25°C, in contrast to cells shifted for 2 h at 37°C, in which GFP-RIO proteins display essentially a nuclear and nucleolar pattern of fluorescence. GFP (C) in the crm1-1 strain at either 25°C or 37°C shows a normal cytoplasmic localization pattern. (D) Sedimentation profiles in a glycerol gradient of GFP-Rio2p, GFP-Rio1p, small-subunit ribosomal protein rpS2, and large-subunit ribosomal protein rpL3. A total extract from a crm1-1 strain carrying plasmid pEV24 (LEU2 pNOP::GFP::RIO1) or pEV71 (ADE2 pNOP::GFP::RIO2) and grown at 37°C was loaded on a 10 to 30% glycerol gradient and processed as described in the legend to Fig. 6.