Identification and purification of a subcomplex containing 5S rRNA, ribosomal proteins rpL5 and rpL11, and assembly factors Rpf2 and Rrs1. (A) The pre-rRNP maturation pathway in Saccharomyces cerevisiae. The 90S pre-rRNP containing 35S pre-rRNA and a subset of ribosomal proteins and assembly factors is converted into 43S and 66S precursor particles, then to mature 40S and 60S ribosomal subunits. Four consecutive 66S pre-rRNPs contain 27SA₂, 27SA₃, 27SB, and 25.5 plus 7S pre-rRNAs, respectively, plus ribosomal proteins and assembly factors. Ribosome biogenesis begins in the nucleolus and continues in the nucleoplasm; final steps occur in the cytoplasm. (B, left) Amounts of Rpf2 or Rrs1 sedimenting as a small complex near the top of gradients increase in the rrp1-1 mutant compared with wild-type cells. Wild-type yeast (JWY7087 and JWY7461) and temperature-sensitive rrp1-1 mutant yeast (JWY7089 and JWY7462) expressing TAP-tagged Rpf2 or Rrs1 were grown at 25°C to 1.5 × 10⁷ cells per milliliter and shifted for 5 h to 37°C. Extracts from the wild-type (top) and rrp1-1 mutant (bottom) strains were fractionated on 7%–47% sucrose gradients, and amounts of Rpf2-TAP or Rrs1-TAP proteins in each fraction were assayed by Western immunoblotting. Fractions containing 43S and 66S preribosomes are indicated. (Right) Amounts of Rpf2 and Rrs1 in whole-cell extracts remain the same in rrp1-1 mutant compared with wild-type cells. (C) Enrichment of the Rpf2-subcomplex in the rrp1-1 mutant compared with other preribosomal proteins. Whole-cell extracts from the RRP1 and rrp1-1 strains were subjected to TAP using Rpf2-TAP or Rrs1-TAP. Copurifying proteins were resolved by SDS-PAGE and stained with silver. Proteins enriched in mutant strains are labeled. (D) Enrichment of proteins in TAP-purified samples does not result from changes in their amounts in whole-cell extracts. TAP-purified samples from the RPF2-TAP strains JWY7087 or JWY7089 (top), and proteins from whole-cell extracts of the same strains (bottom), were subjected to Western blot analysis. Samples on the left in each pair are from wild-type RRP1 cells and those on the right are from the rrp1-1 mutant. Proteins from equal amounts of cells were loaded in each lane. Similar results were obtained with the RRS1-TAP strain (data not shown). (E) The purified Rpf2 subcomplex contains Rpf2, Rrs1, rpL5, and rpL11. Fractions 5–7 pooled from the gradients of rrp1-1 extracts, shown in B, were subjected to TAP purification using either Rpf2-TAP or Rrs1-TAP. Purified proteins were resolved by SDS-PAGE, stained by Coomassie blue, and identified by mass spectrometry. (F) 5S rRNA is present in the purified Rpf2 subcomplex. RNA was extracted from whole-cell extracts (left), affinity-purified samples from untagged strain (middle), and Rpf2 subcomplex purified from gradient fractions 5–7 (right), resolved by polyacrylamide gel electrophoresis, and assayed by Northern blotting using specific oligonucleotide probes. (G) Docking of atomic models of ribosomal proteins and 5S rRNA into a 15 Å resolution cryoelectron microscopy map of yeast 60S ribosomal subunits (Spahn et al. 2001) demonstrates that rpL5 (blue), rpL10 (yellow), rpL11 (red), and 5S rRNA (green) are adjacent to one another in mature 60S ribosomal subunits.

5S rRNA, rpL5, and rpL11 are not recruited into 66S preribosomes in the absence of Rpf2 or Rrs1. Yeast strains GAL-RPF2 (JWY8129), GAL-RRS1 (JWY8132), GAL-RPL5 (JWY8108), and GAL-RPL11 (JWY8112) were grown at 30°C in galactose medium to 3 × 10⁷ cells per milliliter. A second culture of each strain was grown in galactose medium and shifted to glucose medium for 16 h to 3 × 10⁷ cells per milliliter to deplete the respective proteins. Whole-cell extracts were subjected to TAP purification, using Nop7-TAP to isolate preribosomes. (A) Proteins present in the affinity-purified preribosomes were resolved by SDS-PAGE and stained with silver (left), or subjected to Western blot analysis (right). In each sample pair, proteins from cells grown in galactose-containing medium are on the left and those from cells transferred to glucose are on the right. (B) RNA was extracted from each of the TAP-purified samples described above, as well as from wild-type cells (JWY6938), resolved by denaturing gel electrophoresis, and assayed for 5S rRNA or 27SB pre-rRNA by Northern blotting or primer extension.

Depletion of Rpf2, Rrs1, or rpL5 causes 66S preribosomes to accumulate in the nucleus. GAL-RPF2 (JWY7703), GAL-RRS1 (JWY7706), or GAL-RPL5 (JWY7696) strains expressing eGFP-tagged rpL25 were grown at 30°C in galactose medium to 3 × 10⁷ cells cells per milliliter. A second culture of each strain grown in galactose medium was shifted to glucose medium for 17 h to 3 × 10⁷ cells per milliliter. Nuclei were stained with DAPI, and ribosomes containing eGFP-rpL25 were detected by fluorescence microscopy.

Model for recruitment and function of 5S rRNA, RpL5, and RpL11. (Top) In wild-type cells, Rpf2 and Rrs1 help to recruit rpL5, rpL11, and 5S rRNA into 90S preribosomal particles containing 35S pre-rRNA. (Bottom) When components of the subcomplex cannot assemble into 90S particles, 35S, 27SA₂, 27SA₃ pre-rRNAs can be processed efficiently. However, processing of 27SB pre-rRNA is blocked. The abortive pre-rRNPs are released from the nucleolus to the nucleoplasm, and cannot be exported to the cytoplasm. When the subcomplex cannot be assembled, Rpf2 and Rrs1 are unstable.

Direct physical interactions between Rpf2, Rrs1, rpL5, and rpL11. (A) Synthetic [³⁵S]methionine-labeled Rpf2, Rrs1, rpL5, or rpL11 proteins (*) were incubated with GST-Rrs1, Rpf2, rpL5, or rpL11 fusion proteins. (Middle lane) As negative controls, synthetic proteins were incubated with GST beads only. Complexes were eluted from glutathione beads, subjected to SDS-PAGE, and detected by autoradiography. Fifty percent of the input ³⁵S-labeled proteins in each assay (left lane) and 100% of the pull-down (right lane) are shown. (B) Purification of an Rpf2/Rrs1 heterodimer from whole-cell extracts. Whole-cell extract from wild-type strain JWY7087 was subjected to affinity purification using TAP-tagged Rpf2. Prior to TAP purification, half of the extract was incubated with phosphatase inhibitors, which disrupts pre-rRNPs. Purified proteins were resolved by SDS-PAGE, stained with silver, and identified by mass spectrometry. (C) Protein–protein interactions within the Rpf2 subcomplex.

Rpf2 and Rrs1 are unstable upon depletion of each of the Rpf2 subcomplex proteins. Yeast strains GAL-RPF2 (JWY8129), GAL-RRS1 (JWY8114 or JWY8132), GAL-RPL5 (JWY8108 or JWY8109), and GAL-RPL11 (JWY8080 or JWY8112) were grown at 30°C in galactose-containing medium to 3 × 10⁷ cells per milliliter. A second culture of each grown in galactose medium was shifted to glucose medium for 17 h to 3 × 10⁷ cells per milliliter. Proteins in whole-cell extracts were assayed by Western blotting. Samples on the left for each pair are from cells grown in galactose medium and those on the right are from cells grown in galactose medium and shifted to glucose medium.

Each component of the Rpf2 subcomplex assembles into 90S preribosomes. (A) Whole-cell extracts were prepared from an untagged strain (JWY6147) and Rpf2-TAP (JWY7087) and Rrs1-TAP (JWY7461) strains. RNA was extracted from whole-cell extracts (left) and affinity-purified samples (right). Five micrograms of total RNA and 100% of purified RNA were used to assay 35S, 27SA₂, and 27SB pre-rRNAs by primer extension. Amounts of 27SA₃ pre-rRNA were very low and thus are invisible in some lanes. No pre-rRNAs copurified upon mock purification from untagged strains. Note that different oligos were used in reactions for 35S and 27S pre-rRNAs, and longer exposure was done for 35S pre-rRNA. (B) Preribosomes were affinity-purified using TAP-tagged Nop7. Purified pre-rRNPs containing rpL5-HA3 or rpL11 were immunoprecipitated by anti-HA antiserum. Five micrograms of total RNA (left) and 100% of purified RNA (right) were assayed for 35S, 27SA₂, 27SA₃, and 27SB pre-rRNAs by primer extension. Pre-rRNPs from an untagged strain were used as a negative control.