Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing

Abstract

Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.

Figure 4.

Pre-rRNA structure and processing. (A) Schematic diagram of the 35S pre-rRNA showing the processing sites and oligonucleotides used. The 35S pre-rRNA contains the sequences for the mature 18S, 5.8S, and 25S rRNAs separated by the two internal transcribed spacers, and flanked by the 5′-ETS and 3′-ETS. (B) Major pre-rRNA processing pathway.

Figure 1.

rix9-1 ts strain is complemented by RLP7 and accumulates Rpl25p-eGFP in the nucleolus. (A) rix9-1 is complemented by RLP7. Growth properties of rix9-1 cells, an isogenic wild-type strain, and the rix9-1 strain harboring a plasmid containing RLP7 (pRLP7). Serial dilutions of cells plated on YPD plates were incubated for 3 d at 23 or 37°C. (B) rix9-1 cells or the rix9-1 strain harboring a plasmid containing RLP7 (pRLP7) were transformed with Rpl25p-eGFP and grown at 23°C, before shift for 4 h to 37°C and inspection in the fluorescence microscope.

Figure 2.

Rlp7p is homologous to the ribosomal protein L7. (A) Homology with the eukaryotic L7 is divided into three domains: domain I, exclusively eukaryotic; domain II, signature of the L30 super-family of ribosomal proteins conserved in all kingdoms of life; and domain III, present in eukaryotic and archeal proteins. (B) Multiple sequence alignment of Rlp7p with other homologues: L7a (Schizosaccharomyces pombe), archeal L30 (Methanococcus jannashii) and prokaryotic L30 (Escherichia coli). The two RNA-binding motifs (RBD1 and RBD2) are indicated with shaded boxes. Position of the mutation in rlp7-1 is indicated by an arrow. Accession numbers for Rlp7p and homologues are as follows: S. cerevisiae (GenBank/EMBL/DDBJ accession no. Ynl002c), S. pombe (GenBank/EMBL/DDBJ accession no. O60143), M. Jannashii (GenBank/EMBL/DDBJ accession no. P54046), and E. coli (GenBank/EMBL/DDBJ accession no. P02430).

Figure 3.

Rlp7p is associated with 60S precursors and is required for 60S subunit biogenesis. (A) Rlp7p cofractionates with 60S preribosomes. Analysis of polysomal ribosome fractions derived from cells expressing Rlp7p-ProtA. Fractions were subjected to SDS-PAGE and Western blotting using α-ProtA and α-Rpl13p antibodies. The position of 40S and 60S subunits, of 80S ribosomes, and of polysomes is indicated. (B) Polysomal profiles (OD_{260 nm}) after sucrose density gradient centrifugation derived from the rlp7-1 mutant and wild-type strain grown at permissive temperature (23°C) and 4 h after shift to restrictive temperature (37°C). The position of 40S, 60S, and 80S ribosomes, of polysomes and half-mer polysomes is indicated.

Figure 5.

Analysis of pre-rRNA processing in the rlp7-1 strain. (A) Northern analysis of high–molecular weight RNA separated on a 1.2% agarose/ formaldehyde gel (B) Northern analysis of low–molecular weight RNA separated on an 8% polyacrylamide/urea gel. (C) Primer extension analysis. RNA was extracted from cells growing at 23°C (0 h samples) and after transfer to 37°C for the times indicated. Oligonucleotides used are indicated in parentheses.

Figure 6.

Pulse-chase analysis of pre-rRNA processing in the rlp7-1 strain. Wild-type (lanes 1–5) and rlp7-1 (lanes 6–10) strains were labelled with [H³]-uracil for one min or with [H³-methyl]-methionine for 2 min, and then chased with unlabeled uracil (A) and (C) or methionine (B) for the times indicated.

Figure 7.

Ultrastructural localization of pre-60S particles using a ITS2-specific probe in rlp7-1 cells. Wild-type and rlp7-1cells grown at 25 or at 37°C for 4 h were cryofixated and cryoembedded in LR white resin. ITS2 containing pre-rRNAs were detected with a riboprobe conjugated to biotin.

Figure 8.

Rlp7p is located in the subnucleolar GC compartment. (A) Rlp7p-GFP is nucleolar but does not fully overlap with Nop1p. Strain Rlp7p-GFP harboring a plasmid expressing DsRed-Nop1p was grown at 23°C to OD_600nm of 0.5. The DNA-staining dye DAPI was added to the cells 5 min before microscopic inspection of the GFP signal, DsRed, and DAPI in the fluorescence microscope. Cells were also visualized by Nomarski imaging. Shown are also merged pictures as indicated. (B) Rlp7p colocalizes with Nug2p, but does not overlap with the DFC markers Gar1p and Nop1p. Diploid strains, which were constructed by mating of haploid strains (Table I), expressed the following combinations of spectral GFP variants: YFP-Nop1p/Rlp7p-CFP; Nug2p-YFP/CFP-Nop1p; Gar1p-YFP/CFP-Nop1p; Nug2p-YFP/Gar1p-CFP; and Nug2p-YFP/Rlp7p-CFP. Cells grown at 30°C were harvested in the exponential growth phase (OD_600nm 0.5), before the YFP (green; nucleolar I), CFP (red; nucleolar II) and Hoechst (blue) fluorescence signals were detected by fluorescence microscopy. The green and red signals were also merged (merge). The DNA staining and Nomarski pictures are also shown.

Figure 9.

Immunolocalization of Nop1p and Rlp7p-ProtA by EM. Exponentially growing cells were prepared for EM by high-pressure ultrafast freezing and cryoembedding in LR White resin. Nop1p and Rlp7p-Prot A were detected by immunogold labeling with an anti-Nop1 monoclonal antibody and a polyclonal anti–protein A antibody, respectively. Nop1p and Rlp7p-Prot A are codetected with gold particles of 5 (labeled with a red circle) and 10 nm (labeled with a blue circle) in diameter, respectively. Bar, 100 nm.

Figure 10.

Model of Rlp7p function during nucleolar pre-rRNA processing and ribosome assembly. See text for details.