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. 2007 Nov;13(11):1977-87.
doi: 10.1261/rna.553807. Epub 2007 Sep 5.

Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

Hiroko Yamada  1 , Chihiro Horigome, Takafumi Okada, Chiharu Shirai, Keiko Mizuta
Affiliations

Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

Hiroko Yamada et al. RNA. 2007 Nov.

Abstract

We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth.

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Figures

FIGURE 1.
FIGURE 1.
Rrp14p depletion causes a growth defect. (A) Growth curves of W303–1A transformed with pRS414 (WT; open circle) and KM1411 (GAL1–MYC–RRP14; closed square) cultured at 30°C in SCGal medium and shifted to SC medium. The changes in optical density at 600 nm were followed after the shift. The cell cultures were diluted to keep the optical density lower than 1.0. Data are represented as ODt/OD0 on a logarithmic scale, where t is the time in hours after shifting medium. (B) Western blotting of KM1411 (GAL1–MYC–RRP14) cultured at 30°C in SCGal medium, shifted to SC medium, and cultured for the indicated time. Cell extracts from KM1411 (GAL1–MYC–RRP14, lanes 1–6), KM1412 (MYC–RRP14, lane 7) and W303–1A transformed with pRS414 (WT, negative control; lane 8) were subjected to SDS-PAGE. Western blotting using anti-myc antibodies is shown. The positions of size markers are shown on the left. Asterisks indicate nonspecific bands.
FIGURE 2.
FIGURE 2.
Rrp14p depletion causes a defect in assembly of 60S ribosomal subunits. (A) The polysome profiles from W303–1A transformed with pRS414 (WT) and KM1411 (GAL1–RRP14) strains cultured at 30°C in SCGal, shifted to SC, and cultured for the indicated time. (B) The polysome profiles from the KM1421 (rrp14Δ) strain cells cultured at 30°C in SC. The positions of 40S, 60S, and 80S ribosomal particles, and polysomes are indicated. Arrows indicate half-mer polysomes.
FIGURE 3.
FIGURE 3.
Rrp14p depletion causes a defect in 25S rRNA maturation. (A) The processing pathway of 35S pre-rRNA to the mature rRNAs in S. cerevisiae. Northern probes (a), (b), (c), (d), and (e) used in Figure 4 are shown. (B) Pulse–chase analysis of rRNA synthesis in Rrp14p-depleted cells. W303–1A transformed with pRS414 (WT, lanes 1–4) and KM1411 (GAL1–RRP14 lanes 5–8) were cultured at 30°C in SCGal–Met and Trp, shifted to SC–Met and Trp, and cultured for 18 h. Each culture was pulsed with [methyl-3H] methionine for 3 min and chased with nonradioactive methionine for the indicated times. Total RNA prepared from each sample was analyzed by electrophoresis and blotted to a membrane. The lower part of the membrane was probed for U3 snoRNA as a loading marker.
FIGURE 4.
FIGURE 4.
Northern analysis for steady-state level of rRNAs. (A) W303–1A transformed with pRS414 (WT) and KM1411 (GAL1–RRP14) cultured at 30°C in SCGal were shifted to SC and cultured for the indicated times. Total RNA corresponding to 0.5 OD600 of cells was used for Northern blot analysis. [A(a)] Northern blot analysis was carried out using 32P-labeled DNA probes a, b, c, d, and e, which are shown in Figure 3A. [A(b)] Ethidium bromide staining of a denaturing 6% polyacrylamide/8M urea gel is shown. (B) W303–1A (WT) and KM1422 (rrp14Δ) cultured at 30°C in SC medium. Total RNA corresponding to 0.5 OD600 of cells was used for Northern blot analysis. [B(a)] Northern blot analysis was carried out using 32P-labeled DNA probes a, b, c, d, and e, and a probe for U3 snoRNA. [B(b)] Ethidium bromide staining of a denaturing 6% polyacrylamide/8M urea gel is shown. [A(c)] Mature 25S and 18S rRNA levels shown in B(a) were quantified using BAS-2000 and BAS-1800 (Fuji Photo Film Co.), normalized with the U3 level, and the ratio of the radioactivity value of the rrp14Δ strain per that of wild-type strain serves as the percent.
FIGURE 5.
FIGURE 5.
The rrp14Δ cells grow extremely slowly. (A) Construction of rrp14Δ with the HIS3 gene. (B) Tetrad analysis of the RRP14/rrp14Δ ∷HIS3 strain. Three sets of tetrads were cultured on a YPD plate at 25°C for 12 d.
FIGURE 6.
FIGURE 6.
Run-on transcription assays. (A) The rDNA transcription unit. RNA polymerase I transcribes the 35S primary rRNA transcript, whereas RNA polymerase III transcribes the 5S rRNA in the opposite direction. PCR products corresponding to the indicated segments of the 5′ external transcribed spacer (5′ETS), the 25S rRNA, NTS, and the 5S rRNA were cloned into the pTOPO plasmid. (B) Transcription run-on analysis. The wild-type and rrp14Δ strains were cultured, permeabilized, and exposed to α-32P-UTP for 10 min. RNA was extracted and hybridized to the plasmids slot-blotted onto Nitran membrane. Autoradiograph [B(b)] is exposed for longer than autoradiograph [B(a)].
FIGURE 7.
FIGURE 7.
Rrp14p interacts with Utp11p, Faf1p, and Rps16p. Protein–protein interactions were analyzed by the yeast two-hybrid system. Fivefold serial dilutions of cultures of L40 strain cells transformed with lexA binding domain-fused (BD) and Gal4p activation domain-fused (AD) genes were plated on SC–Trp, Leu, and SC–Trp, Leu, His. As the empty vector containing lexA binding domain (pBTM116) exhibits a background level of expression, 1 mM 3-aminotriazole was added to a SC–Trp, Leu, His plate for the lower five combinations.
FIGURE 8.
FIGURE 8.
The rrp14Δ cells exhibit aberrant cell morphology. (A) Calcofluor staining and cell morphology (DIC) of the KM1422 (rrp14Δ) cells. (B) DAPI staining and cell morphology of the KM1422 (rrp14Δ) cells. Bars indicate 5 μm.
FIGURE 9.
FIGURE 9.
The rrp14Δ cells lose polarity for bud site selection. BY4741(WT) and KM1431 (rrp14Δ) haploid cells were grown to a midlogarithmic phase, sonicated for cell separation, and stained with Calcofluor White to visualize bud scars.
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