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Department of Biochemistry, University of California, Riverside, California 92521-0129,1 Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom,2 Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, North Carolina 275993
Received 12 July 2002/ Accepted 13 August 2002
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ABSTRACT |
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INTRODUCTION |
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Previous work has demonstrated that the histone stem-loop was necessary and sufficient to support the translation of reporter mRNAs in animal cells when present at the 3' terminus and, like a poly(A) tail, was functionally dependent on the cap (17). Although SLBP is necessary to mediate the other regulatory aspects associated with the histone stem-loop, its role in translation initiation has not been demonstrated. Moreover, how SLBP might promote efficient translation initiation is unknown.
In this study, we show that SLBP is required for the translation of mRNAs terminating in the histone stem-loop. The stimulatory effect of SLBP can be recapitulated in Saccharomyces cerevisiae cells expressing mammalian SLBP. Genetic analysis indicated that the translational function of SLBP requires eIF4E, eIF4G, and eIF3. SLBP copurified with these initiation factors when they were isolated through their binding either to the 5' cap or to poly(A)-Sepharose. Coisolation studies of SLBP with cap- or poly(A)-associated initiation factor complexes indicated that the association of SLBP with eIF4F required eIF4G and eIF3. Moreover, eIF4G and SLBP coimmunoprecipitate from mammalian cell extract and copurify as recombinant proteins. These data indicate that SLBP is functionally similar to PABP in that it stimulates the translation of histone mRNAs through an interaction with the 5' terminus of the mRNA that is mediated by cap-associated initiation factors.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Functional mRNA half-life analysis. The rate of luciferase protein production was used as a measure of translational efficiency, and the length of time over which luciferase protein continued to accumulate was used to calculate message stability. Following the delivery of each mRNA construct by electroporation, aliquots of cells were removed at time intervals and luciferase assays were performed. The kinetics of luc mRNA translation were determined by following the appearance of protein as measured by enzyme activity plotted as a function of time. Once loaded onto polysomes, translation proceeds at a rate (i.e., the slope of each curve) that is dictated by its translational efficiency and for a period of time that is determined by the stability of the mRNA. The eventual degradation of the mRNA results in a decreased rate of protein accumulation. Following degradation of the mRNA, further accumulation of luciferase protein ceases, represented by the plateau of each curve. Those forms of an mRNA that are more stable will be translationally active longer, represented in a kinetic analysis by a longer period of time over which the protein will continue to accumulate. The functional half-life is defined as the amount of time needed to complete a 50% decay in the capacity of an mRNA to synthesize protein.
m7GTP-Sepharose 4B and poly(A)-agarose purification. eIF4F from yeast and mammalian cells was purified with m7GTP-Sepharose as previously described (47). For yeast, the crude extract was made from spheroplasts (from 250 ml of cell culture) in 2.5 ml of buffer M (20 mM Tris [pH 7.5], 0.2 mM EDTA, 100 mM KCl, 5 mM MgCl2, 5% glycerol, 5 mM ß-mercaptoethanol, and 1 mM dithiothreitol [DTT] with protease and phosphatase inhibitors). Mammalian 293 cells in buffer M were sonicated for 1 min, the cell debris was pelleted, and the supernatant was used for binding to m7GTP-Sepharose 4B. The m7GTP-Sepharose resin was washed three times with buffer M. Two hundred microliters of resin was used per 250 ml of yeast culture, and 100 µl of resin was used for two 100-mm-diameter plates of 293 cells. Binding was carried out at 4°C for 30 min with gentle shaking. The resin was collected, the supernatant was saved as the flowthrough, and the resin was washed three to five times with buffer M and eluted with 100 µM m7GTP.
For poly(A)-agarose purification, cell extract was prepared as described above, except that buffer PA [10 mM HEPES (pH 7.6), 100 mM potassium acetate, 1 mM CaCl2, 1 mM magnesium acetate, 5% glycerol, 1 mM DTT, and protease inhibitors] was used. Purification was carried out as previously described (32). After batch binding for 30 min at 4°C, the resin was collected and washed three times with buffer PA and protein was recovered in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis sample buffer.
Purification of His-tagged SLBP by metal affinity chromatography. Co2+ resin (Clontech) was used as described in the manufacturer's protocol. Cells were disrupted in binding buffer (50 mM sodium phosphate [pH 7.0], 300 mM NaCl) with sonication. Extract was bound to resin at 4°C for 30 min and washed three times with binding buffer (with 5 mM imidazole), and bound protein was eluted with 150 mM imidazole.
Western analysis. Anti-eIF4E and anti-PABP antisera were raised against recombinant protein. A human anti-eIF4A antibody was used to detect the yeast homolog. Anti-SLBP antibody was raised against the C-terminal 13-amino-acid region as described previously (58). Following resolution by SDS-polyacrylamide gel electrophoresis, protein was transferred to a nitrocellulose membrane which was blocked in 5% dry milk-phosphate-buffered saline containing 0.1% Tween 20 (PBST) for 2 h. The membrane was incubated with the primary antibody (used at dilutions of 1:1,000 to 1:2,000) for 1 to 2 h at room temperature, washed three times with PBST, incubated with secondary antibody (immunoglobulin G-horseradish peroxidase) for 1 h at room temperature, and washed three times with PBST, and the signal was revealed by an enhanced chemiluminescence reaction. Membranes were reprobed following stripping by incubating the membrane in buffer D (2% SDS, 62.5 mM Tris [pH 6.8], 100 mM ß-mercaptoethanol) for 15 min at 60°C with shaking.
Immunoprecipitation. Cell extract was prepared as described above with buffer IP (20 mM Tris [pH 7.5], 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 0.1% Triton X-100, 0.1% SDS, and protease and phosphatase inhibitors) and was precleared with protein A/G-Sepharose 4B resin. Antibody (normally 1 to 5 µl of antibody for 2 ml of extract) was added for 4 to 12 h at 4°C. Two hundred microliters (50% slurry) of protein A/G resin was added; the resin was incubated for 2 h at 4°C, collected, and washed three to five times with buffer IP; and the bound protein was used for Western analysis.
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RESULTS |
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The primary and secondary structure of the metazoan histone stem-loop is highly conserved (Fig. 2). These elements include a 6-bp stem consisting of two GC base pairs at the base, a set of three pyrimidine-purine base pairs forming the central portion of the stem and a UA base pair at the top, and a 4-base loop in which the first and third positions are uridines and the fourth position varies. Mutations to the conserved positions of the loop or stem disrupt its 3'-end processing (14, 42) and translational (17) functions and abolish SLBP binding to the histone stem-loop in animal cells (61). To investigate whether the same mutations would be similarly deleterious to the function of the histone stem-loop in yeast expressing SLBP, the expression from luciferase mRNAs terminating in mutant stem-loops (Fig. 2) was examined. Altering the two conserved uridines in the loop to adenosines (luc-SLA1,3) abolished stem-loop function in SLBP-expressing yeast as did inverting either the entire stem-loop (luc-SLR) or just the stem (luc-SLReverse stem) (Table 1). Internalization of the stem-loop (i.e., luc-SLInternal) also resulted in a substantial, although not complete, loss of function in good agreement with the results obtained in mammalian cells (17). These data demonstrate that, as with mammalian cells, the conserved features of the histone stem-loop that are required for binding SLBP (61) are required for SLBP-mediated translational regulation in yeast.
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13C) did not substantially alter SLBP translational function (Fig. 3B). However, deletion of the C-terminal 52 amino acids (52C) or the N-terminal 87 amino acids (87N 13C) significantly reduced SLBP translational function (Fig. 3B). These data indicate that the domains on each side of the RNA-binding domain are required for SLBP translational function either for protein interaction or for correct protein folding. Together, these data demonstrate that SLBP-mediated translation can be recapitulated in yeast and that SLBP and the histone stem-loop represent a two-component regulatory complex that is necessary and sufficient for this function.
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eIF4E is the small subunit of eIF4F in which eIF4E and eIF4A are physically associated with the scaffold protein eIF4G (18, 27, 37). PABP also interacts with eIF4G (55) and copurifies with eIF4F. To determine whether the presence of SLBP in the eIF4F complex affects the composition of this complex, eIF4F was isolated from control and SLBP-expressing yeast on m7GTP-Sepharose in the absence of reporter mRNA. Components of the complex isolated in the absence or presence of SLBP were visualized by Western analysis (Fig. 4C). SLBP copurified with eIF4F on m7GTP-Sepharose and did not alter the amount of eIF4G associated with eIF4E. SLBP copurified with eIF4F even if the extract was pretreated with RNase (data not shown), suggesting that its interaction with eIF4F is RNA independent. SLBP was present in a high ribosomal-salt-wash fraction of yeast ribosomes in the absence of the stem-loop containing reporter mRNA (Fig. 4D), the data supporting the conclusion that SLBP copurifies with eIF4F.
As the association of SLBP with the 5' cap was eIF4E dependent, we used wild-type yeast and strains harboring mutations in key translation initiation factors to investigate the functional interaction between SLBP and the eIF4F complex. Null mutants were employed for those factors that are not essential, e.g., eIF4B or CAF20, whereas Ts mutants or depletion approaches were employed for those factors that have been shown to have an essential role, including eIF4E, eIF4G, eIF4A, eIF2, eIF3, and PABP. For the analysis, capped luc-SLWT and luc-SLR mRNAs were delivered to each mutant in the absence or presence of SLBP. Since the depletion or loss of an initiation factor would be expected to reduce translation nonspecifically, only the loss of the histone stem-loop-mediated increase in luciferase expression would constitute grounds for concluding that the factor concerned is required for the SLBP-mediated stimulation of translation.
Because SLBP binding to m7GTP was lost in the eIF4Ets mutant strain at the nonpermissive temperature (Fig. 4B), we examined whether the translational function of SLBP required eIF4E. Expression from luc-SLWT mRNA in the wild-type eIF4E strain expressing SLBP was 9.2-fold higher than that from luc-SLR mRNA (Table 2). In agreement with the data presented in Fig. 1, no enhancement was observed in the absence of SLBP expression. Similar results were observed at the nonpermissive temperature (Table 2). Expression from luc-SLWT mRNA in the eIF4Ets mutant strain expressing SLBP was 5.4-fold higher than that from luc-SLR mRNA at the permissive temperature (Table 2). However, at 37°C, translation from the luc-SLWT mRNA was preferentially affected, resulting in a substantial reduction in the SLBP-mediated regulation (Table 2). As cell growth at 37°C did not influence the expression of SLBP, these data suggest that SLBP not only coisolates with eIF4E (Fig. 4) but functionally requires this factor.
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We next investigated whether SLBP functionally requires eIF4G. eIF4G is expressed from two genes in yeast, and the proteins (eIF4G1 and eIF4G2) are only 53% homologous (19). Mutant strains expressing only eIF4G1 (tif4632) or eIF4G2 (tif4631) are viable, but the double null is inviable (19). Because the depletion of both isoforms of eIF4G can be achieved by treatment of the yeast with rapamycin (4), we examined the function of SLBP in wild-type or mutant strains expressing only eIF4G1 or eIF4G2 before or after rapamycin treatment. Treatment of yeast with rapamycin results in the rapid degradation of eIF4G without affecting the levels of other eIF4F subunits, i.e., eIF4E and eIF4A (4). Although rapamycin treatment has been reported to affect the transcription of some genes in yeast (45), rapamycin selectively reduces the level of eIF4G through the destabilization of the existing eIF4G protein (4). We confirmed that no degradation of eIF4E (see below) or eIF4A (data not shown) was observed following rapamycin treatment. Moreover, rapamycin treatment did not reduce the level of PABP (see below) or eIF3 (data not shown). Successful depletion of eIF4G following rapamycin treatment was confirmed (data not shown). Expression from luc-SLWT mRNA in the wild-type CW04 strain expressing SLBP was 9.2-fold higher than that from luc-SLR mRNA (Table 3). This SLBP-mediated enhancement was reduced to control levels in cells treated with rapamycin (Table 3) despite the fact that no alteration in the level of SLBP expression was observed (data not shown). The translational enhancement by SLBP was observed in both the eIF4G1-deleted and eIF4G2-deleted strains (Table 3), suggesting that either isoform of eIF4G was capable alone of supporting SLBP function. The lower level of translational enhancement observed in each mutant was reproducible, possibly resulting from reduced levels of total eIF4G. As with the wild-type cells, the translational function of SLBP was virtually lost in the eIF4G1 or eIF4G2 mutant strains following rapamycin treatment (Table 3), confirming that SLBP function is dependent on eIF4G.
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eIF4G functions to recruit eIF3, which in turn is responsible for promoting binding of the 40S ribosomal subunit to an mRNA (31). To determine whether SLBP requires eIF3, a mutant strain containing a Ts mutation in the Prt1 (eIF3b) subunit (22, 39) was employed. Expression from luc-SLWT mRNA in the wild-type eIF3 strain expressing SLBP was 9.1-fold higher than that from luc-SLR mRNA, which was altered little when the cells were shifted to 37°C (Table 4). SLBP-mediated translational enhancement was reproducibly lower in the prt1-1 mutant strain at the permissive temperature and was abolished when the cells were shifted to 37°C (Table 4) despite the fact that no alteration in the level of SLBP expression was observed, the data suggesting that SLBP function is dependent on eIF3.
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subunit of eIF2 (eIF2) at serine 51 by GCN2 in response to amino acid starvation reduces eIF2 activity in S. cerevisiae (28, 29). Mutation of Ser-51 to alanine (eIF2-S51A) prevents phosphorylation and results in constitutive activity, whereas mutation of the serine to aspartic acid (eIF2-S51D) mimics phosphorylation and results in constitutively reduced activity (11). To examine whether changes in eIF2 function affect SLBP function, expression from luc-SLWT and luc-SLR mRNAs was measured in the absence or presence of SLBP in wild-type, GCN2 mutant yeast and strains expressing eIF2 containing either the S51A or S51D mutations (Table 5). SLBP function in these strains was examined in cells not experiencing amino acid starvation (i.e., for histidine) and following starvation for histidine, which induces GCN2 activity. SLBP was expressed to a similar level in all strains (data not shown). In nonstarved cells, translation from luc-SLWT mRNA in wild-type eIF2/GCN2 cells expressing SLBP was 13-fold higher than that from luc-SLR mRNA (Table 5). No reduction in the degree of SLBP-mediated enhancement of translation was observed in eIF2-S51A/GCN2 or eIF2/gcn2 cells (Table 5). Furthermore, in eIF2-S51D/GCN2 cells, the degree of SLBP-mediated enhancement was not reduced (Table 5). In starved cells, the SLBP-mediated enhancement of translation was abolished in eIF2/GCN2 cells as well as in eIF2-S51A/GCN2 and eIF2-S51D/GCN2 cells. A similar loss in SLBP function was observed in stationary-phase cells (data not shown). Partial retention of SLBP activity was observed in eIF2/gcn2 cells, possibly indicating that eIF2 may be partially functionally required for SLBP or that the loss of GCN2 function may lead to changes in other components of the translational machinery that are involved in the activity of SLBP. These data suggest that amino acid starvation results in changes in the translation machinery that result in the loss of SLBP-mediated regulation and are partially controlled by GCN2 but may not involve changes in eIF2 phosphorylation.
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Interaction of SLBP with eIF4F requires eIF4G and eIF3 but not eIF4E or eIF4A. As the genetic analysis in yeast revealed a functional requirement for eIF4E, eIF4G, and eIF3 for the SLBP-mediated regulation of translation and SLBP can be retained on m7GTP-Sepharose (Fig. 4), these data suggest that SLBP may physically interact with one or more of these proteins. Such an interaction may promote a physical interaction between the termini of histone mRNAs, functionally circularizing the mRNA and facilitating its translation. The requirements for the interaction of SLBP with eIF4F were further investigated by examining the binding of SLBP to m7GTP-Sepharose when SLBP was expressed in the same strains described above (eIF4G2 [tif4631], eIF4G1 [tif4632], eIF4Ets, eIF4Ats, or prt1-1). In addition, the requirement for eIF4G was investigated by examining whether SLBP from control or rapamycin-treated tif4631 and tif4632 cells was retained on m7GTP-Sepharose. As observed in Fig. 4, SLBP was retained on m7GTP-Sepharose from extract prepared from tif4631 and tif4632 mutants (Fig. 6). However, retention of SLBP on m7GTP-Sepharose was lost when either mutant was treated with rapamycin (Fig. 6). The effect of rapamycin treatment on reducing the level of eIF4G appeared to be selective, as the treatment had no effect on the level of eIF4E in crude extract (Fig. 6), as has been reported previously (4) as well as on eIF4A, eIF3 (see above), and PABP (see below), suggesting that the reduction in the coisolation of SLBP was not a result of a decrease in eIF4E or these other factors. The observation that SLBP was retained on m7GTP-Sepharose in tif4631 or tif4632 mutants prior to but not following their treatment with rapamycin suggests that the association of SLBP with eIF4F requires eIF4G. Retention of SLBP on m7GTP-Sepharose was observed in the eIF4Ets mutant at the permissive temperature but not at the nonpermissive temperature (Fig. 6), confirming the requirement for eIF4E in mediating the binding of SLBP to m7GTP-Sepharose, observed in Fig. 4. In contrast, the retention of SLBP on m7GTP-Sepharose when expressed in the eIF4Ats mutant was similar at the permissive and nonpermissive temperatures (Fig. 6), suggesting that eIF4A is not required for SLBP to copurify with eIF4F. Retention of SLBP on m7GTP-Sepharose was unaffected in the prt1-1 (eIF3bts) mutant strain at the permissive temperature but not at 37°C (Fig. 6), indicating that active eIF3 is at least partially required for SLBP to copurify with eIF4F.
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Together, the data from yeast and mammalian cells suggest that the physical interaction of SLBP with eIF4F requires eIF4G, and perhaps eIF3, but does not require eIF4E, eIF4A, or PABP.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observations presented in this study suggest that SLBP functionally and physically interacts with eIF4G and eIF3. This conclusion is supported by multiple independent lines of evidence. First, the function of SLBP in yeast was genetically dependent on eIF4G and eIF3 (Tables 3 and 4). Second, copurification of SLBP with the eIF4F/eIF3/PABP complex was observed when the latter was purified by m7GTP-Sepharose or poly(A)-agarose chromatography (Fig. 4C, 6, and 7). Third, eIF4G and eIF3 were required for the association of SLBP with the eIF4F/eIF3/PABP complex (Fig. 7). Fourth, copurification of eIF4G and eIF3 with SLBP was observed when the latter was affinity purified as a His-tagged protein (Fig. 8). Fifth, mammalian eIF4G coimmunoprecipitated with SLBP (Fig. 9B). Sixth, SLBP coimmunoprecipitated with eIF4G or with eIF3 from mammalian cells (Fig. 9C). Seventh, an interaction between recombinant mammalian eIF4G and SLBP was observed (Fig. 9D). The observation that recombinant human SLBP and eIF4G interact in vitro suggests that the interaction may be direct.
In addition to eIF4G and eIF3, eIF4E was necessary for SLBP translational function (Table 2). This finding is consistent with the observation that the translational enhancement conferred by the histone stem-loop structure is cap dependent (17) and that the translational function of SLBP requires a 5' cap (Fig. 4A). Because no direct physical interaction between SLBP and eIF4E was observed (Fig. 9D), eIF4E likely contributes to SLBP function by facilitating the binding of eIF4F to the 5' cap, which may be necessary for the interaction between SLBP and eIF4G in a manner analogous to the role of eIF4E in facilitating the interaction between eIF4G and PABP during the translation of capped and polyadenylated mRNAs (56, 60). Therefore, a model incorporating the present observations posits that the functional interaction of the 5' cap with the 3' histone stem-loop structure involves an interaction of the cap-bound eIF4E with eIF4G, which in turn interacts with the SLBP-bound 3'-terminal histone stem-loop structure (Fig. 10). The presence of other initiation factors, including eIF2 (which may be partially required for SLBP function) and eIF1, e1F1A, and eIF5, would be expected as part of the initiation complex.
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Our results suggest that, from the perspective of translational regulation, SLBP has evolved as a functional mimic of PABP in that it binds to a 3'-terminal sequence, exhibits a functional requirement for the 5' cap, copurifies with the eIF4F complex, and requires eIF4G to mediate its association with the complex. However, SLBP is distinct from PABP in that it may involve an interaction with eIF3 and regulates histone mRNA stability in a cell cycle fashion. Further investigation into the functional and physical parallels between SLBP and PABP in their interaction with the translational initiation machinery will provide valuable insight into the evolution of diverse molecular mechanisms that promote efficient translation.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the United States Department of Agriculture (NRICGP 99-35301-7866) and the National Science Foundation (MCB-9816657) to D.R.G and a grant from NIH (GM29832) to W.F.M. Work in the labs of S.J.M. and V.M.P. is supported by program and equipment grants from The Wellcome Trust (040800, 050703, 045619, and 056778), and S.J.M. is a Senior Research Fellow of The Wellcome Trust.
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FOOTNOTES |
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) mRNAs following their delivery to control yeast (
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