Functional analysis of Saccharomyces cerevisiae ribosomal protein Rpl3p in ribosome synthesis

Abstract

Ribosome synthesis in eukaryotes requires a multitude of trans-acting factors. These factors act at many steps as the pre-ribosomal particles travel from the nucleolus to the cytoplasm. In contrast to the well-studied trans-acting factors, little is known about the contribution of the ribosomal proteins to ribosome biogenesis. Herein, we have analysed the role of ribosomal protein Rpl3p in 60S ribosomal subunit biogenesis. In vivo depletion of Rpl3p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This phenotype is likely due to the instability of early and intermediate pre-ribosomal particles, as evidenced by the low steady-state levels of 27SA(3), 27SB(S) and 7S(L/S) precursors. Furthermore, depletion of Rpl3p impairs the nucleocytoplasmic export of pre-60S ribosomal particles. Interestingly, flow cytometry analysis indicates that Rpl3p-depleted cells arrest in the G1 phase. Altogether, we suggest that upon depletion of Rpl3p, early assembly of 60S ribosomal subunits is aborted and subsequent steps during their maturation and export prevented.

Figure 1.

Pre-rRNA processing in S. cerevisiae. (A) Structure and processing sites of the 35S pre-rRNA. This precursor contains the sequences for the mature 18S, 5.8S and 25S rRNAs that are separated by two internal transcribed spacer sequences, ITS1 and ITS2, and flanked by two external transcribed spacer sequences, 5′ ETS and 3′ ETS. The mature rRNA species are shown as bars and the transcribed spacer sequences as lines. The processing sites and the various probes used are indicated. (B) Schematic representation of the pre-rRNA processing pathway of the 35S pre-rRNA and pre-5S rRNA. Cleavage and trimming reactions are indicated. The data presented in this study suggest that Rpl3p is required for stability of the 27S pre-rRNAs. For reviews on pre-rRNA processing and the known processing enzymes, see (6,8).

Figure 2.

Depletion of Rpl3p results in a deficit in free 60S r-subunits and in the accumulation of half-mer polysomes. Strain JDY511 [YCplac33-RPL3] (RPL3) was grown in YPGalS at 30°C (A) or shifted to YPDS for 24 h (B). Strain JDY511 [pZGA196] (GAL::RPL3) was grown in YPGalS at 30°C (C) or shifted to YPDS for 6 h (D). Cells were harvested at an OD₆₀₀ of 0.8, cell extracts were prepared and 10 A₂₆₀ of each extract were resolved in 7–50% sucrose gradients. The A₂₅₄ was continuously measured. Sedimentation is from left to right. The peaks of free 40S and 60S r-subunits, 80S free couples/monosomes and polysomes are indicated. Half-mers are labelled by arrows.

Figure 3.

Effects of Rpl3p depletion on steady-state levels of pre-rRNAs and mature rRNAs. Strains JDY511 [YCplac33-RPL3] (RPL3) and JDY511 [pZGA196] (GAL::RPL3) were grown in YPGalS medium and then shifted to YPD medium. Cells were harvested at the indicated times and total RNA was extracted. (A) RNA corresponding to equal amounts of OD₆₀₀ units of cells were resolved on a 1.2% agarose–formaldehyde gel, transferred onto a nylon membrane and hybridized consecutively with different probes. (B) RNA corresponding to equal amounts of OD₆₀₀ units of cells was resolved on a 7% polyacrylamide–urea gel, transferred onto a nylon membrane and hybridized consecutively with different probes. Probe names are indicated between parentheses (see Figure 1A for their location in the 35S pre-rRNA).

Figure 4.

Effects of Rpl3p depletion on steady-state levels of 27S pre-rRNAs species. The same RNA samples described in the legend of the Figure 3 were used for primer extension analysis. Probe f (see Figure 1A for its location in the 35S pre-rRNA) was labelled and used for the reactions. Note that this probe allows detection of 27SA₂ (as the stop at site A₂), 27SA₃ (as the stop at site A₃), 27SB and 7S pre-rRNAs (as stops at sites B_1L and B_1S).

Figure 5.

Depletion of Rpl3p leads to nuclear retention of the 60S r-subunit reporter Rpl25p-eGFP. The GAL::RPL3 strain carrying the plasmids pRS315-Rpl25p-eGFP and pRS314-DsRed-Nop1p were grown in SGalS-Leu-Trp medium (Gal+Sorbitol) to early log phase and shifted for 12 h to SDS-Leu-Trp (Glc+Sorbitol). The subcellular localization of the Rpl25p-eGFP and DsRed-Nop1p was analysed by fluorescence microscopy. Triangles indicate the position of the nucleolus.

Figure 6.

Depletion of Rpl3p leads to an arrest of the cell cycle at the G1 phase and an abnormal cell morphology. (A) Cell morphology of GAL::RPL3 cells grown in YPGalS (Gal+Sorbitol) or shifted for 12 h to YPDS (Glc+Sorbitol). Cells were stained with DAPI for localization of nuclei and then visualized by fluorescence and phase contrast microscopy. Merged images are shown. (B) FACS analysis of unsynchronized GAL::RPL3 cells grown in YPGalS or shifted for 12 h to YPDS at 30°C. 1C and 2C peaks correspond to cells with unreplicated and duplicated genomes, respectively.