Yeast strains and plasmids

All yeast strains, plasmids and oligonucleotides used in this study are listed in the S1, S2 and S3 Tables, respectively.

Plasmid pHK1349 was created by amplification of the RPL11B ORF + 900bp upstream of the start codon by the oligonucleotides HK1485 and HK1486 from genomic yeast DNA. The PCR-fragment and pHK12 were digested with SacII and XhoI and subsequently ligated.

pHK653 was digested with XbaI and SacI and the resulting fragment was inserted into pHK87 by using the same restriction sites resulting in pHK666.

pHK1288 was created by digestion of pHK1288 (pGEX-6P-1) and pHK1282 (pGEX-4T-1-DBP5) with EcoRI and XhoI and ligation of DBP5 in the pGEX-6P-1 backbone.

pHK789 was generated by amplification of the ARX1 ORF + 1000bp upstream of the start codon by HK558 and HK562 from genomic yeast DNA and insertion in the pGEM-T plasmid (Promega). The resulting vector was digested with SacII and XhoI and the fragment inserted into the SacII and XhoI digested pHK12.

The strains HKY1356 and HKY1369—HKY1372 were generated by crossing HKY894 with HKY456 and HKY734 with HKY128, respectively.

HKY128 was transformed with pHK629 (2μ RAT8 URA3) and screened for the loss of the CEN rat8-3 LEU2 plasmid to generate HKY456.

The diploid strain Y25036 from Euroscarf was transformed with pHK707 (CEN mtr2-33 LEU2) and after sporulation the haploid spores were selected for Geneticin resistance (mtr2::kanMX4) and the maintenance of the plasmid to obtain the strain HKY734.

The strain HKY90 was created by crossing the strain AG157.206A (rat8-1) [13] into the S288C background.

The strain HKY130 was crossed with the strain rpb1-1 [22] to obtain HKY462.

Growth analyses

Cells were spotted in 10-fold serial dilutions onto different selective agar plates and grown for three days at the indicated semi-permissive temperatures. To allow comparable growth on ura^- leu^- trp^- selective plates, all strains were transformed with plasmids containing the corresponding marker genes and temperature-sensitive alleles were rescued by the presence of the respective wild type genes on URA3 containing plasmids. For growth analyses, cells were spotted onto FOA (5-Fluoroorotic Acid) containing plates to select for the loss of the covering wild type genes. The following strains have been used:

HKY36+pHK86+pHK87+pHK88 (WT), HKY894+pHK87 (nmd3-2), HKY456+pHK638+pHK86 (rat8-3), HKY1356+pHK638 (rat8-3 nmd3-2), HKY456+pHK655+pHK86 (rat8-2), HKY1356+pHK655 (rat8-2 nmd3-2), HKY1370+pHK87+pHK88 (WT), HKY1371+pHK86+pHK88 (rat8-3), HKY1372 (mtr2-33), HKY1369+pHK706 (rat8-3 mtr2-33).

Sucrose-density fractionation experiments

The preparation and fractionation of sucrose-density gradients was essentially carried out following the protocols published previously [20, 23]. Briefly, yeast cell cultures of 200 ml were grown over night at 25°C to log-phase and then shifted for 1 h to the indicated temperatures. Cycloheximide (Roth) was added to a final concentration of 100 μg/ml and cells were incubated for 15 min on ice. After harvesting, the cell pellets were lysed with the same amount of glass beads in a FastPrep-24 machine (MP Biomedical) in lysis buffer (20 mM HEPES-KOH pH7.5, 10 mM KCl, 25 mM MgCl₂, 1 mM EGTA, 1 mM DTT, 100 μg/ml Cycloheximide) supplemented with Complete, EDTA-free protease inhibitor cocktail (Roche). The lysates were centrifuged twice for 10 min at 21000 x g and 4°C. If indicated, the lysates were treated with 0.25 mg/ml RNase A (AppliChem) or with 100 mM EDTA pH 8.0 for 20 min on ice. For protein analyses 10 OD_260nm units, for subsequent co-immunoprecipitations 25 OD_260nm units and for ribosomal profile analyses 7 OD_260nm units of lysates were loaded onto the top of linear 7–47% (w/v) sucrose gradients (20 mM HEPES-KOH pH7.5, 10 mM KCl, 2.5 mM MgCl₂) poured with the Gradient Master machine (Biocomp) and centrifuged for 3 h at 40000 rpm and 4°C in a TH-641 rotor and Sorvall WX80 ultracentrifuge (Thermo Scientific). The gradients were fractionated with a density-gradient fractionator (Teledyne Isco) while the absorbance at 254 nm was documented.

Protein fractions were precipitated with 10% TCA and subjected to SDS-PAGE and Western blotting. To be able to load the whole gradient on one gel, some fractions were pooled as indicated.

For subsequent co-immunoprecipitations, the indicated fractions from three gradients per strain were pooled, diluted with PBSKMT buffer (1x PBS, 3 mM KCl, 2.5 mM MgCl₂, 0.3% Triton-X 100), mixed with 10 μl slurry of BSA-blocked GFP-Trap_A beads (Chromotek) and incubated on a rotator for 4.5 h at 4°C. If indicated, the samples were treated with 0.1 mg/ml RNase A (AppliChem). Afterwards, the beads were washed six times with PBSKMT, mixed with 25 μl 2x SDS-sample buffer and analyzed by SDS-PAGE and Western blotting. As input control, 100 μl of the pooled fractions were precipitated with 10% TCA.

From the profiles of the EDTA treated cells, the 60S/40S ratio was determined from three independent experiments and the Fiji software (1.48s Java 1.6.0_65) was used to calculate the areas underneath the 40S and 60S peaks, respectively.

Western blot analyses

Polyclonal rabbit antibodies against Rpl35 (kindly provided by M. Seedorf, dilution 1:5000), Hem15 and Aco1 (kindly provided by R. Lill, dilution 1:7000 and 1:2000 respectively), Mex67 (kindly provided by C. Dargemont, dilution 1:50000), Rio2 (kindly provided by K. Karbstein, dilution 1:2000), Dbp5 (dilution 1:1000) and Rps3 (dilution 1:1000) were used. GFP-tagged proteins were detected with an anti-GFP antibody (sc-8334; Santa Cruz, dilution 1:1000) and myc-tagged proteins with an anti-myc antibody (sc-789; Santa Cruz, dilution 1:750). Monoclonal mouse antibodies against Pab1 (Santa Cruz, dilution 1:1000) and GST (sc-138; Santa Cruz, dilution 1:2000) and secondary anti-rabbit IgG (H+L)-HRPO and anti-mouse IgG (H+L)-HRPO (Dianova) antibodies were used. The signals were detected with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) and the FUSION-SL chemiluminescence detection system (Peqlab) and the Western blot analyses were quantified using the Bio1D software (Peqlab).

Fluorescence in situ hybridizations (FISH)

The experiments were performed essentially as described [24]. Digoxigenin (DIG)-labeled RNA probes were used for detection of 25S and 18S rRNAs. For probe synthesis, PCR templates with a T7 transcription site on the antisense strand were generated by using HK1138 + HK1139 (25S rRNA probe) and HK1140 + HK1141 (18S rRNA probe). The antisense RNA probes were produced by in vitro transcription of the purified PCR templates with T7-RNA-polymerase (Thermo Scientific) and the DIG-UTP RNA labeling mix (Roche). A Cy3-end labeled oligo(dT)₅₀ probe was used for detection of poly(A)⁺RNAs. If indicated, 25S rRNAs were detected with the Cy3-labeled oligonucleotide HK2200.

Log-phase cells were shifted for 1 h to the indicated temperatures and then fixed with 4% formaldehyde at room temperature followed by a zymolyase treatment. Spheroplasts were permeabilized with P-solution (see below)/0.5% triton X-100 and then pre-hybridized with Hyb-mix (50% formamide, 5x SSC, 1x Denhardts, 10 μg/ml heparin) supplemented with 500 μg/ml of tRNAs and 500 μg/ml of denatured Salmon sperm-DNA for 1 h. Cells were further treated with DIG-labeled RNA probes, Cy3-labeled oligo-25S or Cy3-labeled oligo(dT)₅₀ probes at 37°C overnight followed by washing once with 2x SSC, once with 1x SSC, once with 0.5x SSC at 37°C and once with 0.5x SSC at 25°C (30 min per washing step). DIG was detected with a sheep anti-Digoxigenin Fab-FITC antibody (Roche) diluted 1:200 in ABB (1x PBS, 10% heat-inactivated FCS, 0.3% tween-20). Cell nuclei were stained with Hoechst 33342. Microscopy was performed with a Leica AF6000 microscope and pictures were taken with a Hamamatsu 1394 ORCA-ERA camera and the LAS AF 1.6.2 software (Leica).

A magnification of a representative cell is shown for each strain and the amount of cells revealing similar nuclear accumulation of the fluorescent signal is indicated. The fluorescent signals of the whole cell and of its nucleus of at least 10 cells per strain from at least three independent experiments were quantified by using the Fiji software (1.48s Java 1.6.0_65). Subsequently, the intensity of the nuclear signal was related to that of the complete cell and to the ratio of wild type to calculate the average fold enrichment of the nuclear signal.

GFP-microscopy

Mid-log phase yeast cells were shifted for 1 h to their non-permissive temperatures. Afterwards, the cells were briefly fixed with 2.6% formaldehyde, immediately centrifuged for 5 min at 2050 x g and 4°C and washed with 0.1 M phosphate-buffer and with P-solution (0.1 M phosphate-buffer pH6.5, 1.2 M sorbitol). After transfer on polylysine (Sigma Aldrich)-coated microscope slides, the cells were permeabilized by treatment with 0.5% TritonX-100/P-solution and the DNA was stained with Hoechst 33342. Microscopy studies were performed as described in “Fluorescence in situ hybridizations”.

Immunofluorescence

The experiments were performed as described previously [25]. Yeast cells were grown to mid-log phase, shifted for 1 h to their restrictive temperatures and fixed with 4% formaldehyde for 1 h at room temperature. After zymolyase treatment, spheroplasts were transferred to polylysine-coated microscope slides, permeabilized with 0.5% triton X-100/P-solution (0.1 M phosphate-buffer pH6.5, 1.2 M sorbitol), blocked with antibody blocking buffer ABB (1x PBS, 10% heat-inactivated FCS, 0.3% tween-20) for 1 h at room temperature and incubated with anti-Nop1 antibodies diluted 1:2500 in ABB overnight at 4°C. Following four times washing with ABB, anti-rabbit Alexa Flour 594 secondary antibody (from Invitrogen) diluted 1:100 in ABB was added for 2 h at room temperature. After washing with ABB and 0.1% Tween-20/PBS, the DNA was stained with Hoechst 33342 and microscopy studies were performed as described in “Fluorescence in situ hybridizations”.

For co-localization of 25S rRNAs and Nop1 proteins, the in situ hybridization protocol was performed first with the Cy3-labeled oligonucleotide HK2200 against 25S rRNAs followed by the immunofluorescence protocol with anti-Nop1 primary antibodies and anti-rabbit Alex Fluor 488 secondary antibodies (from Invitrogen).

Northern blotting

DIG-labeled RNA probes against the 25S rRNA (HK1138 + HK1139), 18S rRNA (HK1140 + HK1141), 20S, 33S and 35S pre-rRNAs (HK1893 + HK1894), 27S, 33S and 35S pre-rRNAs (HK1895 + HK974) and U2 snRNA (HK1723 + HK1724) were synthesized as described in “Fluorescence in situ hybridizations”.

Indicated strains were shifted for 1 h to their restrictive temperatures and total RNA from mid-log yeast cells was extracted by using the kit “NucleoSpin RNA” (Macherey-Nagel) following the protocol of the manufacturer. 5 μl of RNA solution in DEPC-water containing 1 μg of total RNA were mixed with 10 μl RNA-Loading Dye (50% deionized formamide, 6% Formaldehyde, 1x MOPS, 25 ng/ml Ethidium bromide, 10% Glycerol, Bromophenol blue and Xylene cyanol), denatured for 10 min at 65°C and electrophoresed on a 1% agarose/MOPS gel containing 2% formaldehyde in running buffer (1x MOPS) at 80V for 4–5 h. After washing the gel once in DEPC-water and twice in 20x SSC for 15 min, the RNA was transferred onto a positively charged nylon membrane (Amersham Hybond-N⁺). RNA was crosslinked by exposing the membrane for 7 min to UV light (5000 mJ) and incubation for 2 h at 80°C. After pre-hybridization for 1 h at 68°C with hybridization buffer (0.5 M Na-Phosphate pH7.2, 7% SDS, 1 mM EDTA), 1 μl of each DIG-labeled RNA probe was added and hybridized overnight at 68°C. The membranes were subsequently washed four times for each 15 min in 2x SSC/ 0.1% SDS at room temperature, 1x SSC/ 0.1% SDS at room temperature and twice in 0.5x SSC/ 0.1% SDS at 68°C. DIG was detected with anti-Digoxigenin-AP, Fab fragments (Roche) and as substrate CSPD (Roche) following the protocol of the manufacturer on X-ray films.

Co-immunoprecipitation experiments

The experiments were performed as described previously [23]. Yeast cells were grown in 300–500 ml selective media to mid-log phase and if required, shifted for 1h to the indicated temperatures. After harvesting, the cell pellets were lysed with the same amount of glass beads in a FastPrep-24 machine (MP Biomedical) in PBSKMT (1x PBS, 3 mM KCl, 2.5 mM MgCl₂, 0.5% Triton-X 100) supplemented with protease inhibitor cocktail for yeast (Sigma-Aldrich) and Complete, EDTA-free protease inhibitor cocktail (Roche). After two times centrifugation for 10 min at 21000 x g and 4°C, 500–1000 μl of the clarified lysate were incubated with 10 μl slurry of GFP-Trap_A beads (Chromotek) for 3 h rotating at 4°C. If indicated, the samples were treated with 0.2 mg/ml RNase A (AppliChem) for 30 min at 4°C. The beads were washed six times with PBSKMT and mixed with 25 μl 2x SDS-sample buffer. After boiling, the eluted proteins were separated on 10% SDS-polyacrylamide gels and analyzed by Western blotting.

In vitro-binding studies

His-Mtr2 and Mex67 were co-expressed in E. coli BL21 DE3 cells and the heterodimer was purified by affinity chromatography with Protino Ni-NTA agarose (Macherey-Nagel) and stored at -80°C in elution buffer (30 mM HEPES pH7.5, 100 mM NaCl, 10% glycerol, 250 mM imidazole). GST-Dbp5 and GST, respectively, were expressed in E. coli Rosetta 2 cells and purified by affinity chromatography with GSTrap 4B Glutathione Sepharose (GE Healthcare) and stored at -80°C in elution buffer (for GST-Dbp5: 50 mM Tris pH 7.5, 150 mM NaCl, 30 mM Glutathione reduced; for GST: 30 mM HEPES pH 6.5, 100 mM NaCl, 10% glycerol, 2 mM DTT, 30 mM Glutathione reduced).

For binding studies, 100 μg of purified GST-Dbp5 or 100 μg of GST were mixed with 100 μl of bacterial lysates and incubated with 10 μl slurry of Glutathione Sepharose 4B beads (GE Healthcare) in lysis buffer (20 mM HEPES pH7.5, 100 mM NaCl, 4 mM MgCl₂, 10% Glycerol, 0.1% NP-40, 1 mM DTT, protease inhibitor Complete from Roche) for 1 h rotating at 4°C. The lysis buffer was supplemented with 1 mM ATP and/or 0.2 mg/ml RNase A (AppliChem) if indicated. The beads were washed once with lysis buffer. Subsequently, 28 μg of purified His-Mtr2+Mex67 was added and incubated for 1 h at 4°C. Afterwards, the beads were washed six times with lysis buffer and bound proteins were eluted with 40 μl of 2x SDS-sample buffer. Half of the eluates and 14 μg of GST-Dbp5 / 16 μg of GST / 9 μg of His-Mtr2+Mex67 as input controls were subjected to SDS-PAGE and Western blotting analyses or Coomassie staining, respectively.

Mutants of DBP5 are defective in the nuclear export of both pre-ribosomal subunits

As expected for its function in translation termination, Dbp5 is associated with polysomes (Fig 1A and 1B and [20]). However, we noticed that Dbp5 is also present in the fractions of both free ribosomal subunits in sucrose-density gradient studies (Fig 1A and 1B) and is weakly, but specifically detectable in immunoprecipitations of the large ribosomal protein Rpl25 from these fractions (Fig 1C and 1D). These results suggest that Dbp5 is already transiently associated with free ribosomal subunits and might have a function on the subunits prior to translation. Considering that this RNA-helicase is necessary for the directional transport of mRNAs into the cytoplasm [12, 13], we investigated if Dbp5 would also be required for the nuclear export of pre-ribosomal subunits. Indeed, fluorescence in situ hybridization (FISH) experiments with fluorescent 25S and 18S rRNA probes revealed that almost all cells of the temperature-sensitive dbp5 mutants rat8-2 and rat8-7 accumulate pre-60S and pre-40S subunits in their nuclei upon shift to their restrictive temperatures (Fig 2A and 2B; for all split channels and merged pictures see S1A and S1B Fig). These defects are comparable with those seen in the established export receptor mutant xpo1-1 (Fig 2D) indicating that Dbp5 is required for the nuclear export of both pre-ribosomal subunits. Interestingly, even though also detectable, the effect is less strong and affects fewer cells in rat8-3, while such defects are only barely visible in rat8-1 cells (Fig 2A and 2B and 2D). Likewise, all of these mutants reveal a nuclear accumulation of GFP-tagged Rpl25 and Nmd3 as established reporter proteins for the large [3] and Rps2-GFP as marker for the small ribosomal subunit [26] mirroring the effect seen with the rRNA probes (S2 Fig). In support of our data, rat8 mutants were identified earlier in a screen for mutants that mislocalize another reporter protein of the large ribosomal subunit, Rpl11-GFP, in their nuclei, however, this initial finding was not further characterized [27]. Together, these results show that Dbp5 is required for the nuclear export of pre-ribosomal subunits.

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Fig 1. Dbp5 is associated with polysomes and both ribosomal subunits.

(A-B) Wild type yeast lysates separated in sucrose-density gradients were fractionated while measuring the absorbance at 254 nm (A_254nm) that results in the displayed profile (A). The corresponding separated protein fractions are shown in Western blot analyses with antibodies against Dbp5 and the ribosomal protein Rps3 (B). The percentage of the total cellular amount of Dbp5 in the non-ribosomal, 40S, 60S, 80S and polysomal fractions is indicated. (C-D) Western blot analyses of Rpl25-GFP immunoprecipitations from gradient fractions of the free ribosomal subunits show co-precipitation of Dbp5. Wild type yeast lysates were separated in sucrose-density gradients and upon fractionation, the free ribosomal subunits containing fractions (red framed area) were subjected to co-immunoprecipitation experiments. The correct fractions were chosen according the flow through photometry profiles displayed in (C). Detection of Mex67 served as positive control and the mitochondrial protein Aco1 served as a negative control.

https://doi.org/10.1371/journal.pone.0149571.g001

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Fig 2. Mutants of DBP5 are defective in the nuclear export of both pre-ribosomal subunits.

Fluorescence in situ hybridization experiments with probes against the 25S rRNA (A), the 18S rRNA (B) and poly(A)⁺RNA (C) reveal a nuclear accumulation of the fluorescent signal in different temperature-sensitive dbp5/rat8 mutants and in a mutant of the karyopherin Xpo1/Crm1 (xpo1-1), which served as positive control, upon 1 h temperature shifts to their indicated non-permissive temperatures. The lower panels of (A) and (B) display a magnification of a representative cell, where the FITC channel was merged with the DNA signal. All single channels and merged pictures are shown in S2 Fig. (WT = wild type). (D) Statistical analyses of (A-C). The upper panel indicates the amount of cells with nuclear accumulation (error bars represent the standard deviation). The lower panel shows the average intensities of the nuclear signal of at least 10 cells relative to the whole cell and compared to wild type. Error bars represent the standard deviation and p-values were calculated by an unpaired students t-test (*** = p<0.001 and ** = p<0.01).

https://doi.org/10.1371/journal.pone.0149571.g002

One might think that the ribosomal export defects could be a consequence of the mRNA export defects. However, the intensities of the defects in ribosomal transport do not correlate with the extent of the mRNA export defects in the different dbp5 mutants (Fig 2C and 2D). While rat8-2 and rat8-7 cells both have very strong ribosomal subunit localization defects, their mRNA export defects differ and particularly rat8-7 has the weakest mRNA transport defect of all tested dbp5 mutants. Vice versa, the ribosomal transport defects seen in rat8-1 and rat8-3 are rather mild, but their mRNA export is strongly impaired. Thus, the missing correlation between mRNA and ribosomal transport defects in all dbp5 mutants suggests that the observed ribosomal mislocalizations are not simply caused by defects in nuclear mRNA export. Furthermore, the onset of both, mRNA and ribosomal export defects is rapid and already detectable after short temperature shifts (S3 Fig and [13]), which further argues for a direct role of Dbp5 in ribosomal transport.

Mutants of DBP5 show no unique defects in ribosomal subunit biogenesis

To rule out that the observed defects in pre-ribosomal subunit export are caused by failures of their biogenesis, the steady state level of the different rRNAs and their precursor molecules was analyzed by Northern blotting in the strongest ribosomal transport mutants rat8-2 and rat8-7 (Fig 2). Indeed, only minor changes such as a slight increase in the 35S pre-rRNA levels and the appearance of an aberrant 23S rRNA are visible (Fig 3A). These variations are comparable to those of the temperature-sensitive mutants rat7-1 and xpo1-1 (Fig 3A and [7, 28, 29]), which have also defects in the nuclear export of both pre-ribosomal subunits and mRNAs [7, 27, 30]. Thus, slight defects in the rRNA processing could be a typical phenotype of nuclear export mutants and might be caused by the nuclear retention of pre-ribosomal particles and/or certain mRNAs.

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Fig 3. Mutants of DBP5 show no unique defects in the biogenesis of ribosomal subunits.

(A) The steady state levels of the different rRNAs and their precursors in the dbp5 mutants rat8-2 and rat8-7 are comparable with those in the export mutants rat7-1 and xpo1-1. Northern blot analyses with DIG-labeled RNA probes targeting the indicated rRNA species and U2 snRNAs (loading control) are shown. Total RNA of the different strains was extracted upon 1 h shifts to their restrictive temperatures and 1 μg was separated by formaldehyde/MOPS-agarose gel electrophoresis. (B) The total amount of 40S and 60S subunits is not altered in dbp5 mutants. Flow through photometry (A_254nm = absorbance at 254 nm) profiles of ultra-centrifuged sucrose gradients from rat8-2 and rat8-7 cells shifted to their indicated non-permissive temperatures and treated with 100 mM EDTA reveal no significant change of their peak sizes and the 60S to 40S subunit ratio in comparison to the wild type. In contrast, rpl10(G161D) served as a positive control of the assay and shows a decreased 60S peak.

https://doi.org/10.1371/journal.pone.0149571.g003

In addition to that, we analyzed whether the production of ribosomal subunits in general is disturbed in the dbp5 mutants and determined their overall amount of 40S and 60S subunits. All lysates were treated with 100 mM EDTA to disrupt the 80S ribosomes and to obtain free ribosomal subunits, which were separated in sucrose-density gradients to receive two distinct peaks representing the 40S and 60S subunits. Both peak areas were calculated and set into relation. As expected, wild type cells reveal a 60S:40S ratio of ~2.3 (Fig 3B) [30], while rpl10(G161D) cells defective in the cytoplasmic maturation of large ribosomal subunits [31] show a reduced 60S peak size and a decreased subunit ratio of 1.55 (Fig 3B). In contrast to the maturation mutant, the ratios of rat8-2 and rat8-7 cells are comparable to wild type. Moreover, the entire amounts of both subunits are unchanged, which is reflected by basically the same peak sizes as in wild type. These results indicate that the biogenesis of ribosomal subunits is not detectably affected in dbp5 mutants. As the ribosomal export defects in rat8-2 and rat8-7 are rather strong (Fig 2A and 2B and 2D), it seems unlikely that potential slight processing defects cause the nuclear accumulation of ribosomal subunits.

Dbp5 genetically and physically interacts with ribosomal export factors

Additional support for a function of Dbp5 in transporting pre-ribosomal subunits came from genetic analyses of rat8-3 and rat8-2 cells. Their combination with mutants of NMD3 and MTR2, which are exclusively defective in the export of large ribosomal subunits, but not in mRNA transport [3, 32], result in synthetic lethality (Fig 4A). Interestingly, even the milder export mutant rat8-3 shows the synthetic growth defect with nmd3-2 and mtr2-33. These findings suggest that the mutations in these proteins might affect the same cellular process: The nuclear export of pre-ribosomal subunits. Remarkably, even though genetic interactions of DBP5 with several mRNA processing and export factors were identified earlier [33–35], the synthetic lethality with ribosomal transport factors has not been uncovered before. Although the synthetic growth defects of the double mutants are quite severe, it remains possible that these effects are influenced by the mRNA export defects of the dbp5 mutants. However, this seems rather unlikely, as for example mtr2-21 mutants with exclusive mRNA transport defects show no strong genetic interaction with nmd3-2, whereas mtr2-33 mutants with exclusive ribosomal transport defects are synthetic lethal with nmd3-2 [32].

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Fig 4. Genetic and physical interactions of Dbp5 and ribosomal export factors.

(A) Dbp5 genetically interacts with the established ribosomal transport factors Nmd3 and Mtr2. The indicated strains were spotted in serial dilutions onto plates either selecting for a covering the respective wild type gene containing plasmid (selective media) or for the loss of this plasmid (FOA = 5-Fluoroorotic Acid). The strains rat8-3 and rat8-2 in combination with nmd3-2 or mtr2-33, which has a specific export defect for ribosomal subunits, show synthetic growth defects after three days at 30°C. (B) Physical interactions of Dbp5 with Nmd3, Xpo1 and Rio2. Western blot analyses show co-precipitated Nmd3-myc in the Dbp5-GFP immunoprecipitation (left panel) and co-precipitated Dbp5 in the Xpo1-GFP and Rio2-GFP pull-downs (right panel). The DBP5-GFP expressing strain and the wild type (WT) were transformed with an NMD3-13xmyc containing plasmid. Detection of Hem15 served as a negative control and of Rps3 as a positive control.

https://doi.org/10.1371/journal.pone.0149571.g004

Thus, to gain further evidence for a role of Dbp5 in ribosomal export, we performed physical interaction studies with established ribosomal export factors. Supportingly, Dbp5 associates with Nmd3-, Rio2- and Xpo1-containing particles in co-immunoprecipitation experiments in vivo (Fig 4B and reciprocal co-immunoprecipitations are shown in S4A and S4B Fig) suggesting that Dbp5 physically contacts the export factor-bound pre-40S and pre-60 ribosomal subunits during nuclear export. Together, these genetic and in particular these physical interactions support a function of Dbp5 in ribosomal transport.

Dbp5 awaits pre-ribosomal subunits at the cytoplasmic side of the NPC

Similar to nuclear mRNA export, Dbp5 might also be needed for ribosomal transport at the cytoplasmic filaments of the NPC to remodel the arriving RNPs. However, it also seems possible that Dbp5 may travel together with the subunits from the nucleus into the cytoplasm. To distinguish between these possibilities, we investigated the localization of Dbp5-GFP in the nmd3-2, mtr2-33 and xpo1-1 strains defective in ribosomal transport (Fig 2A and 2B and [3, 32]). Interestingly, while Dbp5 accumulates in the nuclei of xpo1-1 cells, it remains cytoplasmic in the nmd3-2 and mtr2-33 mutants like in wild type (Fig 5A). These results suggest that Dbp5 is not accompanying pre-ribosomal subunits through the NPC, but as reported earlier is actively exported from the nucleus by Xpo1 [36]. Thus, Dbp5 seems not to shuttle with pre-ribosomal subunits. It might rather act on the subunits from inside the nucleus or from the cytoplasmic side of the NPC.

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Fig 5. Dbp5 does not accompany ribosomal particles through the NPC, but seems to be required at its cytoplasmic side.

(A) Dbp5 does not travel with the pre-ribosomal subunits from the nucleus to the cytoplasm. Fluorescence microscopy images of GFP-tagged Dbp5 show a cytoplasmic distribution in wild type, mtr2-33 and nmd3-2 cells upon shift to 37°C for 1 h. In contrast, GFP-Dbp5 accumulates in the nucleus of shifted xpo1-1 cells. (B) rat7-1 cells show mild ribosomal export defects. Fluorescence in situ hybridization experiments with probes against the 25S rRNA, 18S rRNA and poly(A)⁺RNA are shown in rat7-1 and wild type cells upon shifts for 1 h to 37°C. Statistical analyses were performed as shown in Fig 2D. (C) The association of Nmd3-myc with GFP-Dbp5 is not increased in the drg1-18 strain compared to wild type (WT). Western blot analyses of an immunoprecipitation experiment with GFP-Dbp5 and co-precipitated Nmd3-myc are shown upon 1 h shifts to 37°C. Detection of Hem15 served as non-binding control.

https://doi.org/10.1371/journal.pone.0149571.g005

If Dbp5 is required at the cytoplasmic exit site of the NPC to assist the export of pre-ribosomal subunits, the dissociation of the protein from its cytoplasmic NPC anchor Nup159/Rat7 [36–38] should result in decreased ribosomal transport. Therefore, we performed FISH experiments with rat7-1 cells, in which Dbp5 is detached from the nuclear rim at the restrictive temperature [36]. Indeed, rat7-1 cells show slight, but clearly detectable ribosomal export defects (Fig 5B; for all split channels and merged pictures see S5A Fig). Similar results were obtained for rat7ΔN cells (S5B Fig) specifically lacking the interaction domain for Dbp5 [36]. These defects might not be as strong as the defects seen in dbp5 mutants (Fig 2D), because the presence of untethered Dbp5 in the cytoplasm of the rat7 mutants might be sufficient to support the nuclear export of both pre-ribosomal subunits to some extent. Notably, the export defect of pre-ribosomal subunits present in rat7-1 is considerably milder than that of mRNAs in this mutant, which might indicate differences in both transport pathways. Thus, rather the localization of Dbp5 at the NPC than the function of Nup159 as an ADP-release factor for Dbp5 [17] might be important for the export of pre-ribosomal subunits.

To support ribosomal transport, it is either possible that Dbp5 acts shortly from the cytoplasmic filaments, to which it remains associated, or that the protein is loaded onto the transported ribosomal particles at the NPC. In this case, Dbp5 might not be released until the subsequent cytoplasmic biogenesis steps occur, as described for shuttling maturation and export factors such as Arx1 and Nmd3. They accumulate on immature pre-60S particles in the cytoplasm of a temperature-sensitive mutant of DRG1 (drg1-18), which prevents subsequent maturation steps ([39, 40] and S6 Fig). If this would also be the case for Dbp5, one would expect that the release defects seen in drg1-18 should result in an increased binding of Dbp5 and Nmd3, which is not visible in co-immunoprecipitation studies (Fig 5C). In contrast, the interactions of Mex67 and Nmd3 with Arx1 are enhanced in drg1-18 compared to wild type cells (S6 Fig), showing that the chosen conditions were sufficient to induce cytoplasmic accumulation of immature pre-60S subunits. These findings indicate that Dbp5 is not displaced from ribosomal particles at later cytoplasmic maturation steps, suggesting that the protein acts earlier, immediately upon the cytoplasmic appearance of the pre-ribosomal subunits at the NPC.

The ATPase cycle of Dbp5 seems to be dispensable for ribosomal transport

Localized at the cytoplasmic side of the NPC, Dbp5 was proposed to utilize its ATPase activity for displacement of export factors from emerging mRNAs [15, 17]. To investigate if the regulated ATPase cycle of Dbp5 is also needed for ribosomal transport, we analyzed the impact of ATPase-deficient mutants of DBP5 on ribosomal export. As such mutations are lethal in yeast in the absence of the wild type protein, we overexpressed the ATPase-deficient allele dbp5-R426Q or the mutant dbp5-R369G, which has a reduced ATPase activity, in a DBP5 wild type strain, in which the mutations cause dominant-negative effects on growth and mRNA export [41]. Indeed, overexpression of both alleles does not lead to defects in ribosomal transport, while mRNA export is clearly affected (Fig 6A and 6B) demonstrating that the expression of dbp5-R369G and dbp5-R426Q was efficiently induced. These results suggest that the ability of Dbp5 to hydrolyze ATP might not be required for efficient ribosomal transport. The dominant-negative effect on mRNA export of dbp5-R369G is caused by its competition with wild type Dbp5 for Gle1 [41], which is the ATP-hydrolysis stimulating co-factor of Dbp5 [18, 19]. Thus, Gle1 induced stimulation might not be needed for the function of Dbp5 in nuclear export of pre-ribosomal subunits.

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Fig 6. The ATPase activity of Dbp5 is dispensable for nuclear export of pre-ribosomal particles.

(A-B) Dominant-negative ATPase-deficient dbp5 mutants do not affect ribosomal transport. Fluorescence in situ hybridization experiments with wild type cells overexpressing DBP5, dbp5-R369G and dbp5-R426Q after 1.5 h galactose induction with probes against the 25S rRNA (A) and the 18S rRNA (B) reveal wild typical distribution of the fluorescent signals in contrast to the nuclear accumulations of the poly(A)⁺RNA. (C) The cytoplasmic filament-bound ATPase-deficient mutant dbp5-R426Q partially rescues the ribosomal transport defects of rat8-2. Fluorescence in situ hybridization experiments with Cy3-labled probes against 25S rRNAs are shown in rat8-2 cells with an empty vector (p) or overexpressing dbp5-R369G, dbp5-R426Q, dbp5E240Q, dbp5K144Q and DBP5 after 1 h galactose induction. All cells were shifted for the last 30 min of galactose induction to 37°C. (D) Statistical analyses of (C) show the average enrichment of the nuclear signal compared to rat8-2 cells. The nuclear signals of at least 10 cells were set into relation with the whole cell and the signal obtained in wild type. The resulting ratios were compared to rat8-2. Error bars represent the standard deviation and p-values were calculated by an unpaired students t-test (*** = p<0.001 and * = p<0.05).

https://doi.org/10.1371/journal.pone.0149571.g006

This interpretation is further supported by analyses of different mutants of GLE1. While gle1 mutants have severe defects in nuclear mRNA export (S7E Fig and [42]) emphasizing the importance of functional Gle1 in mRNA export, only few cells slightly accumulate ribosomal rRNAs (S7A and S7B Fig) or ribosomal reporter proteins (S7C and S7D Fig) in their nuclei in accordance to previously published ribosomal export screens [7, 27]. Together, these findings suggest that Gle1 is not required to stimulate the ATPase activity of Dbp5 for ribosomal transport.

To directly analyze the need of the ATPase cycle of Dbp5 for ribosomal export, we overexpressed different ATPase-deficient dbp5 alleles in the temperature-sensitive rat8-2 mutant and found that overexpression of dbp5-R426Q leads to a partial rescue of the ribosomal transport defects in these cells (Fig 6C and 6D). A nuclear accumulation of 25S rRNAs is still visible in these cells, but the average intensity of the nuclear signal compared to the whole cell is decreased to approximately 70% of that of rat8-2 cells, while the mRNA export defect is not reduced (data not shown). The mutated protein dbp5-R426Q has a reduced RNA and ATP binding activity, but is still localized to the nuclear rim and able to bind Nup159 [41]. These findings indicate that rather the positioning of Dbp5 to the cytoplasmic side of the NPC than the ability of Dbp5 to bind RNAs and to hydrolyze ATP might be required for nuclear export of pre-ribosomal particles. In support of this interpretation, the ATPase-deficient dbp5-E240Q and dbp5-K144Q proteins are lost from NPCs and predominantly cytoplasmic [41] and their overexpression does not substantially rescue the ribosomal transport defects of rat8-2 (Fig 6C and 6D). Interestingly, overexpressed dbp5-R369G does also not alter the intensity of ribosomal export defects in rat8-2 cells, although the mutated protein is localized to the nuclear rim. In contrast to dbp5-R426Q, however, this mutant is mainly bound via Gle1 and Nup42 to the NPC [41]. Therefore, only the Nup159-bound conformation of Dbp5 might sufficiently support ribosomal export. In contrast, its ATPase activity seems to be less important, since overexpression of dbp5-R369G, which has a remaining ATPase activity of 60% [41], does not rescue, while NPC-localized overexpressed dbp5-R426Q with only ~10% ATPase activity [41] is able to partially rescue the ribosomal transport defects of rat8-2 (Fig 6C and 6D).

Dbp5 does not displace Mex67 from transported pre-ribosomal subunits

Our results suggest that the localization of Dbp5 to the cytoplasmic filaments of the NPC is crucial for ribosomal transport, whereas its ATPase-dependent helicase activity seems to be dispensable. As a consequence, Dbp5 should not release bound transport factors from ribosomal particles. For mRNA export, in contrast, it was suggested that Dbp5 uses its regulated ATPase cycle to displace export factors such as Mex67 from the emerging mRNAs [14, 15, 17]. Mex67 is also needed for the transport of pre-40S and pre-60S subunits, but when and how the export receptor is released from ribosomal subunits is currently unknown [9, 10]. To address this question, we performed sucrose-density gradient analyses and found that Mex67 is in fact not only part of the fractions that contain the soluble proteins and the ribosomal subunits, which it does during nuclear transport, but it is also bound to polyribosomes in wild type and dbp5 mutants (Fig 7A and S8A Fig). These findings show that Mex67 is part of translating ribosomes and is not dissociated from the ribosomal subunits by Dbp5 immediately after transport. Interestingly, the polysomal association of Mex67 increases in rat8-2 cells (Fig 7A), despite the fact that the defects in mRNA export lead to a reduced rate of translation. This is reflected by a flat polysome profile and a corresponding reduced presence of the ribosomal protein Rps3 and the cap-binding protein Cbp80, which binds mRNAs only in the first rounds of translation [43], in polysomal fractions. This result indicates that Mex67 is not dissociated from mRNAs in the dbp5 mutant. To analyze the ribosome-bound Mex67 pool, we eliminated the mRNA-bound Mex67 molecules through RNase A treatment. RNase A degrades single-stranded RNAs, among them mRNAs engaged in translation, which subsequently destroys the polysomes and leads to one single 80S peak (see profiles of Fig 7B). As shown in Fig 7B and S8B Fig, Mex67 is still detectable in the 80S associated fractions in dbp5 mutants and its amount is similar to that seen in wild type cells. These findings reveal that a pool of Mex67 is in fact always ribosome-bound and not displaced by Dbp5. The mRNA-bound Cbp80 served as a positive control for the successful mRNA degradation, as it is detectable in the polysomal fractions of wild type cells (Fig 7A), but is removed from the ribosomal fractions by the RNase A treatment (Fig 7B).

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Fig 7. Dbp5 does not displace Mex67 from exported ribosomal particles.

(A) Sucrose-density fractionation experiments reveal that Mex67 is associated with polysome-containing fractions from wild type and rat8-2 cells and (B) remains equally ribosome bound upon RNase A treatment. The upper panel shows profiles of wild type and rat8-2 cells upon flow through photometry (A_254nm) from ultra-centrifuged sucrose gradients. Before loading, the cells were shifted for 1 h to 37°C and half of the resulting lysates were treated with RNase A. The bottom panel shows the corresponding separated protein fractions in Western blot analyses with direct antibodies against Mex67 and the ribosomal protein Rps3. The mRNA-binding protein GFP-Cbp80 was expressed with a galactose-inducible promoter, subsequently detected with an anti-GFP antibody to serve as a positive control. The ratios of all proteins from the first fractions (non-ribosomal+40S+60S) and the last fractions (80S+polysomal fractions) are indicated. (C-D) Mex67 is RNA-independently associated with polysomes in wild type cells. Western blot analyses of Rpl25-GFP immunoprecipitations from polysomal gradient fractions show co-precipitation of Mex67. Wild type yeast lysates were separated in sucrose-density gradients that were subsequently fractionated. The polysomes-containing fractions (red framed area) were subjected to co-immunoprecipitation experiments that were treated with RNase A. The correct fractions were chosen according the flow through photometry profile displayed in (C). Detection of Rps3 and Dbp5 served as positive controls. Aco1 was detected as non-binding control and the mRNA-binding protein Pab1 as control for the RNase A treatment. (E) The association of Mex67 with ribosomal proteins is unchanged in the rat8-2 strain. Western blot analyses of a co-immunoprecipitation experiment with Mex67-GFP and Rps3 or Rpl35 in wild type and rat8-2 cells, which were shifted for 1 h to 37°C, are shown upon RNase A treatment. Hem15 served as a negative control.

https://doi.org/10.1371/journal.pone.0149571.g007

To confirm these findings, we immunoprecipitated the large ribosomal protein Rpl25-GFP from polysomes-containing fractions of wild type cells and treated the samples with RNase A. As visible in Fig 7C and 7D, Mex67 can be weakly, but specifically co-precipitated with Rpl25-GFP in contrast to the Poly(A)tail-binding protein Pab1, which is not ribosome bound, but indicates that the RNase A treatment was successful. These findings indicate that Mex67 is at least transiently bound to translating ribosomes in wild type cells.

Together, these results suggest that different pools of Mex67 exist: An mRNA-bound pool that is dissociated by Dbp5 upon transport through the NPC, and a Mex67 pool that remains associated with the exported pre-ribosomal subunits until translation and is not displaced by Dbp5 at the NPC. To further support a model, in which Dbp5 does not displace Mex67 from ribosomal particles, we performed co-immunoprecipitation studies in dbp5 mutants and found that the binding of Mex67 to the ribosomal proteins Rps3 and Rpl35 does not change in rat8-2 cells as compared to wild type (Fig 7E). Vice versa, pulling the GFP-tagged large ribosomal protein Rpl11b results in similar amounts of co-precipitated Mex67 in rat8-2 and wild type strains (S8C Fig). These results show that Dbp5 does not release Mex67 from ribosomal subunits.

Dbp5 directly binds to the export receptor Mex67 in vitro

Our results have uncovered differences in the transport of large RNPs. While the presence of Dbp5 at the cytoplasmic side of the NPC seems to be required for both, mRNA and ribosomal transport, its helicase activity might only be necessary for the nuclear export of mRNAs. This raises the question of how Dbp5 would facilitate ribosomal transport without utilizing its helicase activity. One possibility to support directionality of a transport event is to prevent the backsliding of the export receptor-covered transport cargo by binding the export factors and thus capturing the whole particle at the cytoplasmic side. To investigate if Dbp5 is able to bind Mex67, we analyzed the physical interaction between both proteins in vivo and in vitro. Co-immunoprecipitation studies revealed that Dbp5 and Mex67 mainly associate in vivo via their concomitant binding to single-stranded RNAs (Fig 8A), as reported previously [14]. However, in addition a slight RNA-independent interaction is visible (Fig 8A), which reflects a protein-protein contact. To determine whether both proteins directly interact, we performed in vitro-binding studies with recombinant GST-tagged Dbp5 and the purified export receptor, which is soluble only as a heterodimer, His-Mtr2+Mex67. Indeed, we found a physical interaction between these factors (Fig 8B and S8D Fig), revealing a direct contact between them. Moreover, their binding is even increased in the presence of ATP and is not influenced by the treatment with RNase A. Thus, the enhanced interaction does not depend on the increased affinity of ATP-Dbp5 for RNA [15, 19]. These results indicate that Dbp5 might preferentially bind Mex67 in a nucleotide-bound conformation, which might be the ATP- or ADP-bound state, since Dbp5 could hydrolyze the ATP. One possible model is that this contact might capture the exported pre-ribosomal subunit to the NPC exit and could prevent the back-sliding of the whole particle by covering Mex67 and possibly also other transport factors. This, in addition to the Xpo1-mediated directional transport might carry the ribosomal subunits into the cytoplasm.

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Fig 8. Dbp5 interacts with Mex67 in vivo and in vitro.

(A) Dbp5 interacts mainly RNA-dependently with Mex67 in vivo. Western blot analyses of GFP-Dbp5 immunoprecipitations show strong co-precipitation of Mex67 without RNase A treatment, while addition of RNase A leads to a weaker, but reproducible interaction with Mex67. Detection of Hem15 served as non-binding control. (B) Direct interaction of recombinant GST-Dbp5 and Mex67, which is increased by ATP addition. Western blot analyses of Glutathione Sepharose pull-downs with GST-Dbp5 or GST and the purified heterodimer His-Mtr2 and Mex67 are shown in the presence or absence of 1 mM ATP and 0.2 mg/ml RNase A. The corresponding Coomassie-stained gel is depicted in S8D Fig.

https://doi.org/10.1371/journal.pone.0149571.g008

Taken together, our data uncover differences in the transport mode of Dbp5 for different large RNPs and reveal a novel function for Dbp5 in ribosomal transport, in which it might not require its helicase activity.