The Saccharomyces cerevisiae TIF6 gene encoding translation initiation factor 6 is required for 60S ribosomal subunit biogenesis - PubMed (original) (raw)

The Saccharomyces cerevisiae TIF6 gene encoding translation initiation factor 6 is required for 60S ribosomal subunit biogenesis

U Basu et al. Mol Cell Biol. 2001 Mar.

Abstract

Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit. In Saccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene. To understand the function of eIF6 in yeast cells, we constructed a conditional mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from a repressible GAL10 promoter. Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis. Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, eIF6-depleted cells showed defective pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased 20S pre-rRNA levels, and accumulation of 27SB pre-rRNA. The defect in the processing of 27S pre-rRNA resulted in the reduced formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal subunits in these cells. Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.

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Figures

FIG. 1

FIG. 1

Kinetics of disappearance of 60S ribosomal subunits and inhibition of protein synthesis in eIF6-depleted cells. (A) Exponentially growing cultures of KSY603[GAL10::_Ub-HA-TIF6_] cells in SGal−Met medium were transferred to SD−Met medium and allowed to grow. At the indicated times, cell lysates were prepared from 1 _A_600 unit of cells, and approximately 100 μg of protein from each cell lysate was analyzed by Western blotting using monoclonal anti-HA antibodies as probes. (B) Total ribosomes were isolated from KSY603 (●) and control wild-type (■) cells at indicated times following a shift of each culture from SGal-Met to SD-Met medium. Ribosomes were dissociated into subunits and sedimented through 15 to 40% sucrose gradients as described in Materials and Methods and in reference . (C) Rates of protein synthesis. At the indicated times following shift of cells from SGal-Met to SD-Met medium, 1 _A_600 unit of cells from each culture was harvested and suspended in 300 μl of SD-Met medium containing 0.5 μCi of [35S]methionine (1,175 Ci/mmol), and cells were shaken for 5 min at 30°C. The pulse was then terminated by the addition of a stop buffer containing unlabeled methionine (1.8 mg/ml) and cycloheximide (50 μg/ml). The rate of protein synthesis at each time point was calculated as described previously (29). The data presented in panels B and C are averages of two independent experiments.

FIG. 2

FIG. 2

Synthesis but not stability of 60S ribosomes is affected by eIF6 depletion. Exponentially growing cultures of W303α[_TIF6_] or KSY603[GAL10::_Ub-TIF6_] in SGal−Met medium (approximately 50 _A_600 units of cells) were labeled with 200 μCi of [35S]methionine for 30 min at 30°C. Cells were then transferred to 100 ml of SD-Met medium containing unlabeled methionine (500 μg/ml). At the indicated times, cells (10 _A_600 units) were harvested, washed, and lysed as described in Materials and Methods. Each lysate was subjected to 15 to 40% (wt/vol) sucrose gradient centrifugation to separate the ribosomal subunits and fractionated in an ISCO density gradient fractionator attached to a UA-5 absorbance monitor. The amounts of 60S subunits were quantified from absorbance at 254 nm, while 35S radioactivity content of each fraction was determined by counting in Aquasol in a liquid scintillation spectrometer. The total amount of 35S radioactivity present in either the 40S or the 60S ribosomal subunit pool under each condition following transfer to SD medium was calculated. These values are shown in panel B as the percentage of total counts per minute originally incorporated into 60S or 40S ribosomal subunits prior to transfer to SD medium. The specific radioactivity of 60S ribosomal subunits during growth in SD medium was calculated as counts per minute of 35S radioactivity per 1 _A_254 unit of 60S ribosomal subunits.

FIG. 3

FIG. 3

Schematic diagram of pre-rRNA processing in the yeast S. cerevisiae. (A) Organization and processing sites of 35S pre-rRNA. The 35S precursor contains the sequences for the mature 18S, 25S, and 5.8S rRNAs, which are separated by the internal transcribed spacers ITS1 and ITS2 and two external transcribed spacers, 5′ETS and 3′ETS, at the 5′ and 3′ ends, respectively. The oligonucleotides corresponding to different regions of 35S rRNA that were used as probes in Northern analysis are indicated by numbers 1 through 9. (B) Pre-rRNA processing steps and different processing intermediates. The major cleavage sites, endonucleolytic and exonucleolytic processing steps, processing intermediates, and pathways leading to mature rRNA are indicated (adapted from reference 11).

FIG. 4

FIG. 4

Depletion of eIF6 leads to reduced synthesis 25S and 5.8S rRNAs. (A) Exponentially growing cultures of wild-type strain W303α[_TIF6_] and strain KSY603[GAL10::_Ub-TIF6_] in SGal−Met medium were shifted to SD−Met medium and grown for 90 min to remove eIF6 from KSY603 cells. Cells (40 _A_600 units) were pulse-labeled with 250 μCi of [_methyl_-3H]methionine for 2 min at 30°C and then chased with an excess of unlabeled methionine (500 μg/ml) for the indicated times. Total RNA was isolated from each batch of cells; for each time point, an RNA sample containing about 20,000 cpm of 3H radioactivity was analyzed in a 1.2% formaldehyde-agarose gel and subjected to fluorography as described in Materials and Methods. The positions of mature 18S and 25S rRNAs and pre-rRNAs are indicated. (B) Exponentially growing cultures of KSY603[GAL10::_Ub-TIF6_] and control KSY606[_TIF6_][_URA3_] cells in SGal-Ura medium (_A_600 = 0.5) were transferred to SD-Ura medium and allowed to grow for 90 min, pulsed with 200 μCi of [5,6-3H]uracil for 3 min, and then chased with an excess of unlabeled uracil (1 mg/ml) for the indicated times. Total cellular RNA from each sample was isolated, and approximately 60,000 cpm of each [3H]RNA sample was subjected to 7% polyacrylamide–8 M urea gel electrophoresis, transferred to a Hybond-N+ membrane, and analyzed by fluorography as described in Materials and Methods. The positions of 5.8S and 5S RNAs and 4S tRNA are indicated.

FIG. 5

FIG. 5

Steady-state levels of mature rRNAs and rRNA precursors in eIF6-depleted cells. Haploid yeast strains W303α[_TIF6_] and KSY603[GAL10::_Ub-TIF6_] were grown in YPGal medium and shifted to YPD medium. At the indicated times, cells were harvested, and total RNA was extracted and subjected to Northern analysis using 32P-labeled oligonucleotide probes complementary to different regions of 35S pre-rRNA as described in Materials and Methods and Fig. 3. (A) Probe 9, sequences within the mature 25S rRNA; (B) probe 2, sequences within the mature 18S rRNA; (C) probe 1, sequences upstream of site A0 in the 5′ETS; (D) probe 3, sequences in ITS1 between sites D and A2; (E) probe 5, sequences in ITS1 between sites A3 and B1L; (F) probe 6, sequences within mature 5.8S rRNA; (G) probe 7, sequences in ITS2 between E and C2; (H) probe 6, for detection of small 5.8S rRNA. The positions of mature 25S and 18S rRNAs and different precursor rRNAs are indicated. The blots were also analyzed using a probe corresponding to U1 small nuclear RNA, which served as an internal loading control (not shown).

FIG. 6

FIG. 6

Subcellular distribution of eIF6. (A) Cell fractionation. Lysates of the haploid yeast strain KSY605 expressing a c-Myc-tagged eIF6 from its natural promoter were fractionated into postnuclear supernatant (cytosolic fraction) and nuclear pellet as described in Materials and Methods. Approximately 25 μg of protein from each fraction was subjected to Western blot analysis using rabbit polyclonal anti-c-Myc, anti-yeast eIF5, and anti-Nop1p antibodies as probes. (B) Localization of eIF6-HA fusion protein. Indirect immunofluorescence staining was performed with KSY607 cells expressing eIF6-HA from the TIF6 promoter. Chromatin DNA of yeast cells was stained with DAPI. eIF6-HA was detected by immunofluorescence staining with monoclonal mouse anti-HA primary antibody and Cy3-conjugated secondary antibody (second panel). In the third panel, the photographs of the top two panels were merged. Note that in this merged photograph, the nuclear region assumes a magenta color. In the control panel, indirect immunofluorescence was performed with untagged eIF6 expressed from the TIF6 promoter in KSY606 cells.

FIG. 6

FIG. 6

Subcellular distribution of eIF6. (A) Cell fractionation. Lysates of the haploid yeast strain KSY605 expressing a c-Myc-tagged eIF6 from its natural promoter were fractionated into postnuclear supernatant (cytosolic fraction) and nuclear pellet as described in Materials and Methods. Approximately 25 μg of protein from each fraction was subjected to Western blot analysis using rabbit polyclonal anti-c-Myc, anti-yeast eIF5, and anti-Nop1p antibodies as probes. (B) Localization of eIF6-HA fusion protein. Indirect immunofluorescence staining was performed with KSY607 cells expressing eIF6-HA from the TIF6 promoter. Chromatin DNA of yeast cells was stained with DAPI. eIF6-HA was detected by immunofluorescence staining with monoclonal mouse anti-HA primary antibody and Cy3-conjugated secondary antibody (second panel). In the third panel, the photographs of the top two panels were merged. Note that in this merged photograph, the nuclear region assumes a magenta color. In the control panel, indirect immunofluorescence was performed with untagged eIF6 expressed from the TIF6 promoter in KSY606 cells.

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