Role of the yeast Rrp1 protein in the dynamics of pre-ribosome maturation

Abstract

The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necessary for biogenesis of 60S ribosomal subunits. Processing of 27S pre-ribosomal RNA to mature 25S rRNA is blocked and 60S subunits are deficient in the temperature-sensitive rrp1-1 mutant. We have used recent advances in proteomic analysis to examine in more detail the function of Rrp1p in ribosome biogenesis. We show that Rrp1p is a nucleolar protein associated with several distinct 66S pre-ribosomal particles. These pre-ribosomes contain ribosomal proteins plus at least 28 nonribosomal proteins necessary for production of 60S ribosomal subunits. Inactivation of Rrp1p inhibits processing of 27SA(3) to 27SB(S) pre-rRNA and of 27SB pre-rRNA to 7S plus 25.5S pre-rRNA. Thus, in the rrp1-1 mutant, 66S pre-ribosomal particles accumulate that contain 27SA(3) and 27SB(L) pre-ribosomal RNAs.

FIGURE 1.

The pre-rRNA processing pathway in Saccharomyces cer-evisiae. The 35S primary pre-rRNA transcript containing 18S, 5.8S, and 25S sequences flanked by transcribed spacers is produced by RNA polymerase I. The 5S pre-rRNA is separately transcribed by RNA polymerase III. Processing at the highlighted sites is mediated by several groups of endoribonucleases and exoribonucleases (not shown). Note that there are both major (27SB_S) and minor (27SB_L) pathways for production of 27SB pre-rRNA and subsequent processing to produce 25S plus 5.8S rRNA.

FIGURE 2.

Amino acid sequence alignment of Rrp1p homologs. The program MacVector 6.5.3 (Oxford Molecular) was used to align Saccha-romyces cerevisiae Rrp1p with Homo sapiens Nop52, M. musculus NNP-1, Drosophila melanogaster NNP-1, Caenorhabditis elegans C47E12.7, Arabidopsis thaliana AAL84969, and Schizosaccharomyces pombe SPBC9B6.07. Residues are colored according to their side-chain properties: acidic, red; basic, blue; hydrophobic, white; hydrophilic, green. A consensus sequence is indicated. Note that S. cerevisiae Rrp1p ends at F278 and therefore is considerably smaller than its metazoan homologs.

FIGURE 3.

Rrp1p-GFP localizes to the nucleolus. Fluorescence microscopy was performed to detect Rrp1p-GFP in yeast strain JWY6155 (RRP1-GFP). (A) Fluorescence detected from Rrp1p-GFP. (B) DNA stained with 4′,6 diamidino-2-phenylindole (DAPI). (C) Superimposition of Rrp1p-GFP and DAPI signals. (D) Nomarski differential interference contrast image of the cells. (Pseudocolor was used to visualize Rrp1-GFP, green, and DAPI, red.)

FIGURE 4.

Growth of yeast cells is inhibited upon depletion of Rrp1p. (A) Strains JWY6148 (GAL-RRP1) and JWY6149 (RRP1) were streaked onto galactose- and glucose-containing solid YEP media and incubated at 30°C for 2 or 3 d, respectively. (B) Growth curve of JWY6148 (GAL-RRP1) and JWY6149 (RRP1) on shift from YEPGal liquid medium into YEPGlu liquid medium at 30°C; optical cell densities at 610 mm (Odt) were logarithmically plotted relative to the density of the starting culture (Odo) over time.

FIGURE 5.

The rrp1-1 mutation or depletion of Rrp1p causes a deficiency of free 60S ribosomal subunits. (A) Analysis of ribosomal subunits, ribosomes and polyribosomes in strains JWY6101 (RRP1, left) and JWY6102 (rrp1-1, right): Strains grown at 23°C were shifted to 37°C for 5 h. (B) Strains JWY6149 (RRP1, left) and JWY6148 (GAL-RRP1, right) were grown in YEPGal medium at 30°C (top) and shifted to YEPGlu medium at 30°C for 17 h (bottom). Whole-cell extracts were prepared, and ribosomes and polyribosomes were separated on 7%–47% sucrose velocity gradients. Peaks representing 40S and 60S ribosomal subunits and 80S monoribosomes are labeled; half-mer polyribosomes are indicated by arrows.

FIGURE 6.

Processing of pre-rRNAs is perturbed in the rrp1-1 mutant or on depletion of Rrp1p. (A) Sequences within the pre-rRNAs and the mature rRNAs complementary to the oligonucleotide probes are indicated. (B,C) Depletion of Rrp1p or the rrp1-1 mutation affects the steady-state levels of mature rRNA and pre-rRNA. Strains JWY3400 (RRP1, left) and JWY6102 (rrp1-1, right) were grown at 23°C or shifted to 37°C for 5 h (left). Strains JWY6149 (RRP1, left) and JWY6148 (GAL-RRP1, right) were grown in YEPGal at 30°C and shifted to YEPGlu at 30°C for up to 17 h. RNA was extracted, resolved on agarose or acrylamide gels, and detected by (B) Northern hybridization or (C) primer extension. U2 snRNA and U3 snoRNA were used as loading controls. (D) The rrp1-1 mutation disrupts kinetics of pre-rRNA processing and blocks accumulation of 25S rRNA. Strains JWY6101 (RRP1) and JWY6102 (rrp1-1) were grown at 23°C, shifted to 37°C for 5 h, and pulse-labeled with[5,6⁻³H]-uracil for 10 min at 37°C. After the pulse, excess cold uracil was added and equal volumes of cells were collected for RNA extraction at different times of the chase. RNAs were resolved by gel electrophoresis and detected by autoradiography.

FIGURE 7.

AP-tagged Rrp1p cosediments with 66S pre-ribosomes on sucrose velocity gradients. Whole-cell extracts were prepared from JWY6144 (RRP1-TAP) and fractionated on 7%–47% sucrose velocity gradients. Peaks representing 40S and 60S ribosomal subunits and 80S ribosomes are labeled. Fractions from the sucrose gradients were collected, and proteins were TCA-precipitated and subjected to immunoblot analysis to detect Tap-tagged Rrp1p.

FIGURE 8.

Rrp1p associates with pre-rRNAs in vivo. Whole-cell extracts were prepared from the RRP1-TAP strain JWY6144, and the untagged RRP1 control strain JWY3400. RNA was extracted from whole cells and from the affinity-purified samples from both tagged and untagged strains. (A) RNAs were detected using Northern blotting with specific oligonucleotide probes complementary to pre-rRNA and mature rRNAs. (B) Primer extension was used to detect A₂, A₃, and B_L and B_S 5′ ends (27S plus 7S) as well as 25.5S and 35S pre-rRNAs. One hundred percent of TAP affinity-purified and 5% of total RNA were assayed.

FIGURE 9.

Rrp1p associates with ribosomal proteins and nonribo-somal proteins necessary for production of 60S ribosomal subunits. Whole cell extracts were prepared from the RRP1-TAP strain JWY6144 and subjected to tandem affinity purification. Affinity-purified Rrp1p-TAP fractions were precipitated, resolved on a 4%–20% SDS-PAGE gel, and stained with Coomassie blue. Polypeptide bands were excised and identified by mass spectrometry (Tables 1 ▶, 2 ▶).