Yeast shuttling SR proteins Npl3p, Gbp2p, and Hrb1p are part of the translating mRNPs, and Npl3p can function as a translational repressor

Abstract

A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysome-associated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP.

FIG. 1.

Significant amounts of SR-type shuttling mRNA binding proteins associate with polysomes. (A) Extracts of wild-type strains expressing either Cbp80-GFP, GFP-MTR10, NAB2-GFP, MEX67-GFP, GBP2-GFP, HRB1-GFP, or PAB1-GFP were fractionated through 15 to 50% sucrose gradients and subjected to Western blot analysis. Absorbance at 254 nm shows the distribution of ribosomes (top). The corresponding fractions (bottom) were separated on SDS-9% polyacrylamide gels, and the proteins of interest were detected by Western blotting using either anti-GFP, anti-Hrp1p, anti-Npl3p, or anti-Rps3p antibodies. Quantification of the amount of all proteins tested in nonribosomal fractions (left of the gray line) and in the ribosomal fractions (right of the gray line) is shown on the right. (B) Quantification of three independent experiments shown in panel A. (C) Rapid sedimentation of Npl3p, Gbp2p, and Hrb1p requires polysome integrity. Extracts of wild-type cells expressing GBP2-GFP or HRB1-GFP were either mock treated (top) or treated with puromycin (bottom) for 15 min at 22°C to disrupt polysomes prior to sucrose gradient sedimentation. Because puromycin activity requires progression through an elongation cycle, the elongation inhibitor cycloheximide was omitted, and GTP was added. The arrows indicate the monosome fractions that are increased in puromycin-treated cells (two arrows) because of the disruption of the polysomes. Quantification of the protein amounts in the polysome fractions (right of the gray line) is indicated on the right.

FIG. 2.

Deletion of SKY1 has no influence on the dissociation of Npl3p from polysome-associated mRNAs. (A) Mislocalization of Npl3p is similar in mtr10-. and npl3-27 strains and less pronounced in SKY1 deletion strains. Wild-type sky1::TRP1 and mtr10-. cells expressing NPL3-GFP and npl3-27 carrying either an empty vector or 2μm MTR10 were grown in uracil-free medium to log phase. Half of the cultures were retained at 25°C, and the other half were shifted to 37°C for 30 min before they were fixed and analyzed either directly by fluorescence microscopy or indirectly by immunofluorescence using anti-Npl3p antibodies. (B) The absence of Sky1p has no influence on the amount of Npl3p associated with polysome containing mRNAs. The sky1::TRP1 strain and its isogenic wild-type strain were grown in YPD medium to log phase before extracts were fractionated through 15 to 50% sucrose gradients and subjected to Western blot analysis with anti-Npl3p and anti-Rps3p antibodies. Quantification of the amount of both proteins in nonribosomal fractions (left of the gray line) and in the ribosome-associated fractions (right of the gray line) is shown on the right. The experiment shown represents a typical result from three independent experiments.

FIG. 3.

Mtr10p is involved in the dissociation of Npl3p from polysome-associated mRNAs. (A) The mtr10-. strain is defective in the dissociation of Npl3p from the polysome containing mRNA. The mtr10-. strain and its isogenic wild-type strain were grown in YPD medium to log phase and shifted to 37°C for 30 min before extracts were fractionated through 15 to 50% sucrose gradients and subjected to Western blot analysis with anti-Npl3p and anti-Rps3p antibodies. Quantification of the amount of both proteins in nonribosomal fractions (left of the gray line) and in the ribosome-associated fractions (right of the gray line) is shown on the right. The experiment shown represents a typical result from three independent experiments. (B) The mtr10-. strain has no mRNA export defect after a 30-min temperature shift. Samples of log-phase cells were shifted to 37°C for 30 min or 1 h before samples were analyzed by in situ hybridization experiments with Cy3-labeled oligo(dT)₅₀ probes. (C) New synthesis of a reporter protein is significantly reduced in the mtr10-. strain. Wild-type and mtr10-. cells carrying a galactose inducible GFP reporter plasmid were grown in raffinose-containing medium to log phase at 25°C. Cells were then shifted to 37°C for 15 min before galactose was added to induce the production of the reporter gene. Equal samples with OD₆₀₀s of 1.0 were lysed after 0, 5, 10, and 20 min of induction and analyzed by Western blotting using anti-GFP antibodies.

FIG. 4.

The cytoplasmic mislocalization of Gbp2p in sky1Δ cells does not lead to an increased association of the protein with polysomes. (A) Wild-type and sky1::TRP1 cells expressing NPL3-GFP and GBP2-GFP were grown in URA-free medium to log phase at 25°C before they were fixed and analyzed by fluorescence microscopy. (B) The absence of Sky1p has no influence on the amount of Gbp2p associated with polysome containing mRNAs. The sky1::TRP1 strain and its isogenic wild-type strain carrying GBP2-GFP were grown in URA-free medium to log phase before extracts were fractionated through 15 to 50% sucrose gradients and subjected to Western blot analysis with anti-GFP and anti-Rps3p antibodies. Quantification of the amount of both proteins in nonribosomal fractions (left of the gray line) and in the ribosome-associated fractions (right of the gray line) is shown on the right. The experiment shown represents a typical result from three independent experiments.

FIG. 5.

The npl3-27 mutant has no mRNA export defect, but npl3-27p is defective in dissociation from polysome-associated mRNAs. (A) The npl3-27 mutant has no mRNA export defect. The rat7-. mutant, encoding a defective nucleoporin required for mRNA export, and the npl3-27 mutant were grown to log phase in YPD medium and then shifted to 37°C for 30 min and 3 h. Cells were fixed and subjected to in situ hybridization experiments using a Cy3-labeled oligo(dT)₅₀ probe. (B) npl3-27p is enriched in the polysome fractions, and this effect is abolished by high-copy-number MTR10. The npl3-27 strain and its isogenic wild-type strain were grown in YPD medium, and npl3-27 cells carrying either an empty vector or MTR10 on a 2μm URA3 plasmid were grown in URA-free medium to log phase and shifted to 37°C for 1 h before extracts were fractionated through 15 to 50% sucrose gradients and subjected to Western blot analysis with anti-Npl3p and anti-Rps3p antibodies. Quantification of the amount of both proteins in nonribosomal fractions (left of the gray line) and in the ribosome-associated fractions (right of the gray line) is shown on the right.

FIG. 6.

The npl3-27 strain exhibits defects in translation. (A) New synthesis of a reporter protein is significantly reduced in the npl3-27 strain. Wild-type and npl3-27 cells containing a galactose-inducible GFP reporter plasmid were grown in raffinose-containing media to log phase at 25°C. Cells were then shifted to 37°C for 30 min before galactose was added to induce the production of the reporter that was analyzed by Western blotting (A) and RT-PCR (B) after the indicated times. (C) RNA degradation of the reporter is not inhibited in npl3-27 cells. Wild-type and npl3-27 cells containing a galactose (Gal) inducible GFP reporter plasmid were grown in raffinose-containing media to log phase at 25°C. Cells were then shifted to 37°C for 30 min (−Gal lane) before galactose was added for 5 min to induce the production of the reporter (+Gal lane). The synthesis was subsequently blocked by the addition of glucose (Glc), and further samples were collected and subjected to RT-PCR analysis after the indicated times. (D) npl3-27 cells contain less total protein. Wild-type and npl3-27 cells were grown to log phase, split into two equal portions, and either retained at 25°C or shifted to 37°C for 3 h. Equal samples with OD₆₀₀s of 0.6 were lysed in SDS sample buffer, and proteins were separated on an SDS-12% polyacrylamide gel that was stained with Coomassie blue (left panel). Total protein amounts of both strains were calculated from three independent experiments and are shown in a graph (right panel). (E) Overexpression of MTR10 suppresses the reporter gene expression defects in the npl3-27 strain. Wild-type and npl3-27 cells carrying a galactose inducible NAB2-GFP reporter plasmid and 2μm MTR10 or an empty vector were grown in raffinose-containing medium to log phase at 25°C. Cells were then shifted to 37°C for 30 min before galactose was added to induce the production of the reporter gene. All samples (in panels A, B, and C) have an OD₆₀₀ of 1.0 for Western blot analyses using anti-GFP antibodies. For the RT-PCR, primers that amplify GFP and endogenous NAB2 or ACT1 were used.

FIG. 7.

The npl3-27 strain has no obvious defects in transcription, rRNA synthesis, and nonsense-mediated decay. (A) The npl3-27 strain has no transcriptional defect. Northern blot hybridization of total poly(A)⁺ RNA of wild-type, npl3-27, and rpb1-. cells. Cells were grown to log phase at 25°C before they were shifted to 37°C for 30 min. Equal samples with OD₆₀₀s of 2.5 were lysed, and total RNA was isolated, spotted onto a nylon membrane, and hybridized with a ³²P-labeled poly(dT) probe. (B) The npl3-27 strain has no obvious defects in rRNA synthesis. Cells were grown to log phase at 25°C before they were shifted to 37°C for 30 min. Equal samples with OD₆₀₀s of 8.0 were lysed, and total RNA was isolated and separated on a 1% agarose gel. (C) The npl3-27 strain is not impaired in nonsense-mediated mRNA decay. Wild-type, npl3-27, and upf1 strains expressing a nonsense-containing mini-PGK1 gene were grown to log phase and shifted to 37°C for 30 min. Total cellular RNA was isolated, and the amounts of the endogenous PGK1 mRNA (loading control) and the accumulation of the mini-PGK1 mRNA were determined by Northern blot analysis.

FIG. 8.

npl3-27p suppresses growth defects caused by the presence of translation elongation inhibitors or by a mutation in eIF5A. (A) Ten-microliter yeast suspensions with similar cell numbers were spotted in 10-fold serial dilutions onto YPD medium and YPD medium containing 0.1 μg of cycloheximide/ml or 1 mg of paromomycin/ml. The npl3-27 strain was spotted in the top lanes, and its isogenic wild type was spotted in the bottom lanes. (B) Growth of serial dilutions of wild-type and tif51A mutant cells is shown on YPD plates containing cycloheximide where indicated. The strains were carrying either an empty vector (bottom lanes) or a plasmid containing npl3-27 under the strong ADH1 promoter (top lanes). All plates were incubated for 3 to 5 days at the indicated temperatures.