Diazaborine treatment of yeast cells inhibits maturation of the 60S ribosomal subunit - PubMed (original) (raw)
Diazaborine treatment of yeast cells inhibits maturation of the 60S ribosomal subunit
Brigitte Pertschy et al. Mol Cell Biol. 2004 Jul.
Abstract
Diazaborine treatment of yeast cells was shown previously to cause accumulation of aberrant, 3'-elongated mRNAs. Here we demonstrate that the drug inhibits maturation of rRNAs for the large ribosomal subunit. Pulse-chase analyses showed that the processing of the 27S pre-rRNA to consecutive species was blocked in the drug-treated wild-type strain. The steady-state level of the 7S pre-rRNA was clearly reduced after short-term treatment with the inhibitor. At the same time an increase of the 35S pre-rRNA was observed. Longer incubation with the inhibitor resulted in a decrease of the 27S precursor. Primer extension assays showed that an early step in 27S pre-rRNA processing is inhibited, which results in an accumulation of the 27SA2 pre-rRNA and a strong decrease of the 27SA3, 27SB1L, and 27SB1S precursors. The rRNA processing pattern observed after diazaborine treatment resembles that reported after depletion of the RNA binding protein Nop4p/Nop77p. This protein is essential for correct pre-27S rRNA processing. Using a green fluorescent protein-Nop4 fusion, we found that diazaborine treatment causes, within minutes, a rapid redistribution of the protein from the nucleolus to the periphery of the nucleus, which provides a possible explanation for the effect of diazaborine on rRNA processing.
Figures
FIG. 1.
Schematic diagram of the pre-rRNA processing pathway in S. cerevisiae. The processing of the primary transcript in the 3′ ETS results in formation of the 35S pre-rRNA. Two subsequent cleavages at positions A0 and A1 in the 5′ ETS generate the 5′ end of the 18S rRNA. The precursors of the rRNAs for the small and large subunits are separated by cleavage at A2. Cleavage of the 20S pre-rRNA at site D generates the mature 18S rRNA. Processing of 27SA2 occurs by two alternative pathways. Whereas in the major pathway cleavage at A3 is followed by exonucleolytic trimming to position B1S, the minor pathway creates a different 5′ end, resulting in the 27SB1L precursor, which is longer by 7 nucleotides. Cleavage at C2 splits the 27SB pre-rRNAs into a 7S pre-rRNA and a 25.5S pre-rRNA. 3′-5′ and 5′-3′ exonucleolytic trimming of these precursors produces the mature 5.8S and 25S rRNAs, respectively.
FIG. 2.
Treatment with diazaborine leads to a reduced level of 60S subunits. After incubation of the wild-type strain W303 and the diazaborine-resistant mutant ESY212 in the presence of the inhibitor for different periods of time, extracts were prepared and 6.5 _A_260 units each was loaded on 15 to 50% sucrose gradients as described in Materials and Methods. After ultracentrifugation for 2.5 h at 200,000 × g polysome profiles were collected. (A) W303 and ESY212 incubated for 30 min in the presence and absence of diazaborine. Arrows point to the free 40S and 60S subunit peaks to highlight the massive changes in the treated wild-type strain. (B) Time course of W303 incubated for 30, 60, and 120 min in the presence and absence of diazaborine. Free 40S and 60S subunits and half-mer polysomes observed after 120 min of treatment are indicated by open and filled arrows, respectively.
FIG. 3.
Metabolic labeling with [35S]methionine. The wild-type strain W303 was grown in SD complete medium lacking methionine to early log phase (_A_600 of 0.6), and 13 μCi of the radioactive tracer was added. Diazaborine was added immediately (squares), 10 min (triangles), or 20 min (circles) before addition of [35S]methionine. The untreated control is indicated by diamonds. Incorporated radioactivity was measured by the filter binding assay after different incubation periods. One representative experiment of three independent repeats is shown.
FIG. 4.
Diazaborine blocks processing of the 27S pre-rRNA to the 7S pre-rRNA. Strains W303 and ESY212 were incubated in the presence of diazaborine for 0, 15, and 60 min. RNA was extracted, and sequences complementary to oligonucleotides 001, 002, and 003 were detected by Northern hybridization. (A) Hybridization with probe 001, complementary to the 5′ region of ITS2. (B) Hybridization with probe 002, which binds to the 5.8S rRNA.(C) Hybridization with probe 003, which hybridizes to the 5′ region of ITS1. “(LE)” denotes the panel with a section of a longer exposure showing the D/A2 signal. (D) Schematic diagram of the 35S precursor rRNA with an enlarged view of the ITS1 and ITS2 region. Oligonucleotide probes (001, 002, and 003) used for hybridization are indicated as short bars.
FIG. 5.
Treatment with diazaborine inhibits formation of the 25S rRNA. Cells from the strains W303 and ESY212 were pulse-labeled with [14C]uracil for 2 min and chased with a 50-fold excess of nonradioactive uracil for 0, 3, 15, and 30 min in the presence or absence of 5 μg of diazaborine/ml. RNA was extracted by the hot phenol method. The RNA was separated on 1.2% formaldehyde agarose gels to monitor the processing of high-molecular-weight rRNA precursors. The gel was blotted, and the radioactivity was detected by phosphoimaging.
FIG. 6.
Treatment with diazaborine inhibits formation of the 7S pre-rRNA and the 5.8S rRNA. Cells from the strains W303 and ESY212 were pulse-labeled with [14C]uracil for 2 min and chased with a 50-fold excess of nonradioactive uracil for 0, 3, 15, and 30 min in the presence or absence of 5 μg of diazaborine/ml. RNA was extracted by the hot phenol method and separated on 8% acrylamide-8 M urea gels to detect the formation of low-molecular-weight RNAs. The gel was dried, and the radioactivity was detected by exposure of an X-ray film.
FIG. 7.
Diazaborine inhibits cleavage at position A3 of the 27S pre-rRNA. Strains W303 and ESY212 were treated with diazaborine, and RNA was extracted and subjected to primer extension analysis with oligonucleotide 001. (A) The positions of the primer extension stops A2, A3, B1L, and B1S are indicated by arrows. W303 was incubated for 0, 15, 30, and 60 min and ESY212 was incubated for 0, 15, and 60 min with diazaborine. (B) Sections of an autoradiogram containing the A2 and the B1L and B1S stops that was exposed only briefly to demonstrate the actual extent of the decrease of B1 relative to A2.
FIG. 8.
Short-term treatment with diazaborine does not affect electrophoretic mobility or steady-state levels of NME1 RNA and snR30 snoRNA. Cells from the strains W303 and ESY212 were incubated with diazaborine for 0, 15, and 60 min. The same Northern blot as that shown in Fig. 4 was hybridized with probes specific for NME1 RNA and snR30 snoRNA.
FIG. 9.
Diazaborine leads to mislocalization of GFP-Nop4. The wild-type strain W303 or the diazaborine-resistant DRG1-1 mutant ESY212 expressing the GFP-Nop4 fusion was grown to the early log phase and treated with diazaborine. (A) W303 treated for 10 and 120 min with 5 μg of diazaborine/ml. Each field is represented by a fluorescence view along with that of Nomarski optics. (B) W303 cells treated with 5 μg of diazaborine/ml for 60 min were fixed as described in Materials and Methods, stained with DAPI, and viewed with the FITC and DAPI filters. (C) ESY212 was treated for 10 and 120 min with 5 μg of diazaborine/ml. (D) ESY212 cells treated with 5 μg of diazaborine/ml for 60 min were fixed, stained with DAPI, and viewed with the FITC and DAPI filters.
FIG. 10.
Long-term diazaborine treatment results in accumulation of Rpl7A-YFP, YFP-Drs1, and YFP-Nop7 in the nucleolus. The localization of GFP and YFP fusion proteins in untreated and diazaborine-treated wild-type and mutant cells was detected by fluorescence microscopy. Cells were treated with 5 μg of the inhibitor/ml. (A) W303 and ESY212 cells expressing Rpl7A-YFP were treated for 30 min or 2 h with diazaborine. DAPI staining of fixed cells is also shown. (B) YFP-Drs1 and YFP-Nop7 fusions expressed in W303 and ESY212. (C) For the Mtr4-GFP and Nmd3-GFP fusions only examples of the wild-type cells are shown, but the fusion proteins exhibited the same localization in the resistant mutant. (D) Cells with the empty plasmid pUG36 expressing GFP alone and cells expressing a nuclear localization signal-GFP fusion (pNLS) treated with diazaborine served as controls. Cells shown in panels B to D were treated for 120 min with the inhibitor.
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