Nuclear export competence of pre-40S subunits in fission yeast requires the ribosomal protein Rps2

Abstract

Ribosome biogenesis is an evolutionarily conserved pathway that requires ribosomal and nonribosomal proteins. Here, we investigated the role of the ribosomal protein S2 (Rps2) in fission yeast ribosome synthesis. As for many budding yeast ribosomal proteins, Rps2 was essential for cell viability in fission yeast and the genetic depletion of Rps2 caused a complete inhibition of 40S ribosomal subunit production. The pattern of pre-rRNA processing upon depletion of Rps2 revealed a reduction of 27SA(2) pre-rRNAs and the concomitant production of 21S rRNA precursors, consistent with a role for Rps2 in efficient cleavage at site A(2) within the 32S pre-rRNA. Importantly, kinetics of pre-rRNA accumulation as determined by rRNA pulse-chases assays indicated that a small fraction of 35S precursors matured into 20S-containing particles, suggesting that most 40S precursors were rapidly degraded in the absence of Rps2. Analysis of steady-state RNA levels revealed that some pre-40S particles were produced in Rps2-depleted cells, but that these precursors were retained in the nucleolus. Our findings suggest a role for Rps2 in a mechanism that monitors pre-40S export competence.

Figure 1.

Establishment of a conditional strain of S. pombe for the essential rps2 gene. (A) Tenfold serial dilutions of wild-type (rps2⁺) and rps2-null (rps2Δ; FBY136) cells that express plasmid-borne Rps2 from the nmt1 promoter were spotted onto EMM plates with (right) or without (left) thiamine. (B) Rps2-null cells that express plasmid-borne Rps2 under the control of the nmt1 promoter were grown to early log-phase, and shifted to medium containing (lanes 5–8) or not containing (lanes 1–4) thiamine for 0–12 h. Total cell extracts were prepared and analyzed by immunoblotting using antibodies specific to Rmt1 (upper panel), the 40S ribosomal protein S2 (Rps2; middle panel) and the 60S ribosomal protein L7 (rpL7; lower panel). (C) OD plot showing growth inhibition of FBY136 following addition of thiamine (0 h) to the culture medium.

Figure 2.

Depletion of fission yeast Rps2 results in a deficit in 40S ribosomal subunits. Strain FBY136 was grown to early log-phase before being treated (C–F) or not treated (A and B) by the addition of thiamine to the culture media for 0 h (A and C), 4 h (D), 8 h (E) and 12 h (B and F). Cell extracts were prepared under low-Mg²⁺ ion concentration (40 mM EDTA) that causes all cellular ribosomes to dissociate into separate subunits. Eight A₂₅₄ units of each extract were resolved on 5–45% sucrose gradients and the absorbance (A₂₅₄) was continuously measured. The peaks corresponding to the 40S and 60S ribosomal subunits are indicated in (A and C).

Figure 3.

Pre-rRNA processing in yeast. The RNA polymerase I-dependent 35S precursor is first cleaved at site A₀ to produce the 33S precursor, which is rapidly processed at site A₁ to generate the 32S precursor. The 32S transcript is endonucleolytically cleaved at site A₂ to produce the 20S and 27SA₂ pre-rRNAs. The 20S RNA is exported to the cytoplasm for final processing at site D to generate the mature 18S rRNA. The 27SA₂ precursor can be processed via two alternate pathways as indicated to generate the mature 5.8S and 25S rRNAs.

Figure 4.

Inhibition in 18S rRNA synthesis in Rps2-depleted cells. Strain FBY136 was grown at 25°C in EMM medium to early log-phase and then treated (lanes 6–10) or not (lanes 1–5) with thiamine for 6 h. The cells were then pulse-labeled with [³H]-methionine for 8 min and chased with an excess of unlabeled methionine. Total RNA was extracted from cells samples harvested at the indicated time points and resolved on a 1.25% agarose–formaldehyde gel. The position of the rRNA species is indicated on the right.

Figure 5.

Depletion of Rps2 delays pre-rRNA processing at site A₂ and leads to the accumulation of 20S pre-rRNA. (A) Structure of the ribosomal DNA locus. The ribosomal DNA encodes for the mature 18S, 5.8S and 25S rRNAs. The 18S–5.8S and 5.8S–25S rRNAs are interspaced with internal transcribed spacers 1 and 2 (ITS1 and ITS2), respectively. The 18S–5.8S–25S rRNAs are embedded into noncoding 5′- and 3′-external transcribed spacers (5′-ETS and 3′-ETS). Pre-rRNA processing sites are indicated as uppercase letters (A₀–E) and oligonucleotide probes used for northern blotting are indicated as 1–8. (B) Strain FBY136 was grown to mid-log phase before being treated (lanes 5–8) or not (lanes 1–4) with thiamine. Equal amounts of RNA that were extracted from cell samples collected at the indicated time points (in hours) were separated on agarose–formaldehyde gels and transferred to nylon membranes for northern hybridization. The membranes were hybridized with specific oligonucleotides probes shown in (A) that are indicated to the left. rRNA species are indicated to the right.

Figure 6.

Pre-40S ribosomal subunits accumulate in the nucleolus of Rps2-depleted cells. (A) FISH of pre-rRNA in strain FBY136 that was treated (d–i) or not treated (a–c) with thiamine for 6 h. Pre-rRNA was visualized with a probe complementary to the D-A₂ segment of the ITS1 (panels a, d and g). DNA was labeled with DAPI (panels b, e and h) to visualize the nucleoplasm. (B) The GFP-tagged 40S ribosomal protein S7 (Rps7) was expressed in wild-type (panels c and d) and FBY136 (panels a and b) cells that were previously treated (panels b and d) or not treated (panels a and c) with thiamine for 6h before being visualized for GFP localization by live microscopy. White and red arrowheads indicate the nucleolus and nucleoplasm, respectively.

Figure 7.

Function of Rps2 in fission yeast pre-rRNA processing. Our results indicate that Rps2 is required for efficient processing at site A2. In conditions of Rps2 insufficiency, the 32S precursor is cleaved at site A3, as suggested by the detection of 21S rRNA precursors. The kinetics of pre-rRNA accumulation as determined by pulse-chase assays suggest that most 40S precursors are rapidly degraded in the absence of Rps2. Whether 21S- or 20S-containing particles, or both, are subject to this rapid turnover remains to be determined. The remaining 20S-containing small subunit precursors are not processed into mature 18S rRNA in the cytoplasm because they are retained in the nucleolus.