The region between aa 85 and the RBD is not required for translation.
Our initial experiments had defined a 15-aa region of xSLBP1 (aa 68 to 83) required for translation and demonstrated that the amino-terminal 68 aa and C-terminal 57 aa were not essential for translation (24
). Using an MS2-SLBP fusion protein, Gorgoni and coworkers demonstrated that aa 1 to 127 fused to MS2 had the same stimulatory activity on histone mRNA translation as the full-length protein (7
). We had not tested whether there might also be a role for aa 83 to 126, located between the translation activation region and the start of the RBD. We created mutants in SLBP to address whether this region also plays a role in translation using Xenopus
The assay used to measure the activity of the stem-loop reporters (24
) is diagrammed in Fig. . Control oocytes were injected with buffer or with mRNA encoding xSLBP2, which is inactive in translation. Briefly, oocytes were injected with synthetic capped mRNAs encoding the proteins of interest, incubated for 16 h to allow protein expression, and then injected with the reporter mRNAs, luciferase mRNAs ending in either a stem-loop (Luc-SL) or a tetraloop which does not bind SLBP (Luc-TL), or a poly(A) tail [Luc-poly(A)]. Sixteen hours later, the oocytes were harvested and the luciferase activity was measured. The activity of the Luc-TL was used as a control, and its activity was set at 1. The levels of the expressed proteins were determined by Western blotting. Note that there is some xSLBP1 in the oocyte, and thus oocytes injected with buffer show a small activation of translation of the Luc-SL reporter (24
) (Fig. ).
FIG. 1. Analysis of activity of deletion mutants of xSLBP1 in Xenopus oocytes. (A) Schematic of Xenopus oocyte system used as an in vivo translation assay (24). The structures of the luciferase reporters Luc-SL, Luc-TL, and Luc-poly(A) are shown. Abbreviations: (more ...)
To determine whether the region between the translation activation domain and the RBD of SLBP is required for translation, we created the SLBP mutants shown in Fig. . We deleted the region between aa 112 and 126 (xSLBP1Δ112-126) and also that between 89 and 126 (xSLBP1Δ89-126). We replaced aa 1 to 88 of xSLBP2 with the same region of xSLBP1 (xSLBP1 1-89-xSLBP2), which resulted in a fusion protein that contained just the first 89 aa from xSLBP1. We then deleted the region between aa 89 and the RBD in this clone to give xSLBP2Δ89-126 (x1 1-89). To determine whether a specific sequence was required at positions 89 to 126, we replaced aa 89 to 126 of xSLBP1 with those from xSLBP2 [xSLBP1-(x2 89-126)].
Deletion of the 37 aa from 89 to 126 (xSLBP1Δ89-126) abolished translation activation activity in Xenopus oocytes, while deletion of only 14 aa (the serine-rich linker region) from 112 to 126 (xSLBP1Δ112-126) had no effect on translation (Fig. ). However, when the 37 aa from xSLBP2 were substituted for aa 89 to 126 of xSLBP1 [xSLBP1-(x2 89-126)], the resulting protein had complete activity, even though there is no similarity between xSLBP1 and xSLBP2 in this region. This result demonstrates that the effect of the deletion is likely to alter the spacing between the translation activation region and the RBD and that specific sequences in this region are not required for translation. Similarly, a protein that contains only the first 89 aa of xSLBP1 followed by the remainder of the xSLBP2 protein (xSLBP1 1-89-xSLBP2) had full activity in translation (Fig. ), while a protein that has aa 89 to 126 deleted from this protein [xSLBP2Δ89-126 (x1 1-89)] was inactive. All of these proteins were overexpressed in the oocytes as assayed by Western blotting using xSLBP1 antibody (Fig. ).
These results demonstrate that specific sequences in xSLBP1 between aa 83 and 126 were not required for translation, but an appropriate spacing between the RBD and the translation activation region was important for translation activation. Since we have previously shown that the removal of the first 67 aa of xSLBP1 and the entire C-terminal domain has no effect on translation activity (24
), we conclude that the only essential region for translation lies between aa 68 and 83.
Identification of critical residues in the translation activation region of SLBP.
Comparison of the SLBP sequences from vertebrates reveals that there is extensive conservation of much of the SLBP sequence throughout the protein (33
). However, when we included sea urchin (22
) and Ciona intestinalis
(sea squirt) in the comparison, then the similarity among the SLBPs is only in the RBD and the region we identified as important for translation. The sequences in this region for human, Xenopus
, sea urchin, and Ciona
are compared in Fig. . Extending the comparison to more evolutionarily distant organisms (Drosophila melanogaster
and Caenorhabditis elegans
) results in a loss of obvious similarity among the SLBPs other than in the RBD, and the Drosophila
SLBP is not active in translation in Xenopus
oocytes (Sanchez and Marzluff, unpublished). The sequence comparison suggested that a consensus core sequence required for translation might be DW
E, with the invariant amino acids underlined.
FIG. 2. Identification of amino acids in the translation activation region of SLBP required for activity. (A) The sequences of xSLBP1, hSLBP, sea urchin SLBP (suSLBP), and Ciona SLBP (cSLBP) are compared in the region just before the RBD. The numbering is for (more ...)
Based on this analysis, we made a number of point mutants in the consensus sequence in xSLBP1 and tested them for activity in translation. We assayed the ability of the mutant xSLBP1s to enhance translation of a luciferase reporter mRNA ending with a histone stem-loop in Xenopus
) by use of the experimental system shown in Fig. . Synthetic mRNAs encoding xSLBP1 mutants were injected into stage VI oocytes to express the SLBP. Control oocytes were injected with buffer or with mRNA encoding xSLBP2, which binds the stem-loop but is inactive in translation. Oocytes were then injected with the Luc-SL or Luc-TL reporter mRNA and 16 h later assayed for luciferase activity (Fig. ). The expression of all the proteins was monitored by Western blotting (Fig. ).
Point mutations in the conserved residues reduced activity of xSLBP1 in this assay (Fig. ). Mutation of aspartic acid to alanine (D72A), tryptophan to phenylalanine or alanine (W73F and W73A), valine to glycine or alanine (V77G and V77A), or glutamic acid to alanines (E78A, E79A, and EE7879AA) abolished the translation stimulation activity of SLBP. In contrast, mutation of the nonconserved amino acids glycine and serine to alanines (GS7475AA) did not affect the ability of SLBP to enhance translation of the stem-loop reporter mRNA. These results demonstrate that each of the conserved amino acids is essential in this system. All of the mutant proteins were expressed in excess of endogenous xSLBP1 in Xenopus oocytes (Fig. ), although the D72A and V77A mutants were expressed at lower levels than the other proteins.
Identification of a factor that binds the region of SLBP required for translation activation.
We performed a yeast two-hybrid screen with hSLBP lacking the last 27 aa as the bait (the last 27 aa contains an intrinsic trans
-activation activity), against a HeLa cell cDNA library. One of the clones isolated, hSLIP1, encoded a 222-aa protein that has not previously been functionally characterized (Fig. ). Comparing this clone with the expressed sequence tag (EST) database as well as the mouse genome and EST databases suggested that it encoded a full-length polypeptide plus an extensive portion of the 5′ untranslated region which remained in frame with the initiator AUG. An antibody prepared to the C-terminal peptide of hSLIP1 interacts with a single polypeptide in HeLa cells, which is identical in mobility to the polypeptide produced from the cloned SLIP1 in reticulocyte lysates (Fig. ). Although there are potential alternative spliced forms of SLIP1 in the EST databases, we have no evidence for any alternatively spliced forms of SLIP1 expressed in significant amounts (Fig. ). The sequence of SLIP1 (Fig. ) is most similar to a number of translation factors. The structure of the zebrafish orthologue of SLIP1 has recently been determined in a structure genomics project (PDB 2I2OA and 2I2OB), and the protein contains a number of HEAT repeats which are similar to those found in the middle domain of eIF4G and CBP80 (17
FIG. 3. SLIP1 interacts with the translation activation domain of SLBP. (A) The predicted protein sequence of the SLIP1 protein (accession number EU287989). (B) MS2 viral coat protein (lane 1), hSLIP1 (lane 2), or hSLBP (lane 3) was synthesized by in vitro translation (more ...)
We confirmed the interaction of SLBP and SLIP1 and mapped the region of SLIP1 and SLBP that interact both by directed yeast two-hybrid experiments and by in vitro binding experiments using GST pulldown assays. xSLBP1 interacts with hSLIP1 in a yeast two-hybrid assay (Fig. ). A mutant of xSLBP1 which has aa 68 to 123 deleted (xSLBP1Δ68-123), including the region from 68 to 83 required for translation, did not interact with hSLIP1. Similarly, xSLBP1W73A, which contains a point mutation in the conserved tryptophan 73 critical for xSLBP1 function in translation, also did not interact with hSLIP1, although the deletion and mutant proteins were expressed at levels similar to those seen for the wild-type protein (Fig. , lanes 2 to 4).
We tested the ability of GST-hSLIP1 expressed in E. coli to pull down 35S-labeled xSLBP1 labeled by in vitro translation. The xSLBP1 bound to GST-hSLIP1 (Fig. , lane 3). Binding of the D72A, W73A, E78A, and EE7879AA mutants of xSLBP1 to hSLIP1 was much lower than seen for the wild type and similar to binding to the GST control (Fig. , lanes 6, 9, 18, and 24), and the W73F mutant bound less strongly than wild-type xSLBP1 (Fig. , lane 12). The GS7475AA and E79A mutants bound with efficiencies similar to that of wild-type xSLBP1 in this assay (Fig. , lanes 15 and 21). These results are in agreement with the directed yeast two-hybrid data.
GST fusion proteins containing various N-terminal deletions of hSLBP were used in a reciprocal GST pulldown assay with 35S-labeled hSLIP1 (Fig. ). Both the full-length hSLBP and the hSLBPΔ1-68 protein bound hSLIP1 with equal efficiency (Fig. , lanes 2 and 3), while hSLBPΔ1-81 and hSLBPΔ1-126, which have the entire translation activation domain deleted, bound very weakly to hSLIP1 (Fig. , lanes 4 and 5). Purified recombinant His-tagged hSLBP also interacted with purified recombinant GST-hSLIP1 in vitro, demonstrating that there was no dependence on other proteins or RNAs for this interaction (Fig. ).
hSLIP1 cooperates with SLBP to stimulate translation of a reporter mRNA ending in the histone stem-loop in Xenopus oocytes.
To test whether hSLIP1 plays a role in the translation of histone mRNAs, we injected synthetic mRNAs encoding xSLBP1, hSLBP, or hSLIP1 and we compared the effect of expressing SLBP with the effect of coexpressing SLBP and hSLIP1 on the activation of the Luc-SL reporter. xSLBP1 stimulated expression of the Luc-SL more than hSLBP did, as previously reported (7
). The expression of hSLIP1 alone had little effect on the expression of the Luc-SL reporter (Fig. ), suggesting that in the presence of limiting amounts of xSLBP1 in the oocyte, xSLIP1 is not limiting. However, the expression of hSLIP1 together with xSLBP1 resulted in an additional 1.5- to 2-fold increase in total activity (Fig. ). Moreover, the expression of hSLIP1 together with hSLBP also resulted in an increase in activity, up to the same level as seen for xSLBP1, with hSLIP1 (Fig. ).
FIG. 4. Activation of translation of reporter mRNAs ending in a histone stem-loop by SLIP1. (A) Xenopus oocytes were injected with buffer or with capped synthetic mRNA encoding the indicated protein(s), 16 h prior to the injection of Luc-SL or Luc-TL mRNA. Results (more ...)
We further tested the effect of SLIP1 on the translation of a polyadenylated luciferase reporter mRNA [Luc-poly(A)]. While the expression of hSLIP1 together with xSLBP1 stimulated the translation of Luc-SL compared with what was seen for injection of xSLBP1 alone, hSLIP1 had no effect on the translation of the Luc-poly(A) mRNA (Fig. ). Thus, the effect of SLIP1 is specific for the Luc-SL mRNA.
Western blots confirmed that the hSLIP1 (Fig. ) and SLBP (Fig. ) proteins were expressed in the oocytes and that protein levels were not affected by the injection of reporter mRNAs or the injection of multiple mRNAs. We interpret the low activity level of hSLBP in oocytes compared to that seen for xSLBP1 to the fact that hSLBP may not interact as well with xSLIP1 as xSLBP1 does. We also confirmed that the expression of SLBP or SLIP1 had no effect on the stability of the reporter RNAs in the oocytes, as found previously for SLBP (24
) (Fig. ). We conclude that SLIP1 together with SLBP stimulates the translation of mRNAs ending in the histone 3′ stem-loop.
The translation of a luciferase reporter ending in an MS2 stem-loop is stimulated by an MS2-SLBP fusion protein both in reticulocyte lysates (24
) and in Xenopus
). We tested the ability of an MS2-SLIP1 fusion protein to activate the translation of the luciferase reporter in reticulocyte lysates by use of the approach previously described (24
). Expression of the hSLBP, hSLIP1, and MS2 fusion proteins and luciferase reporters were visualized by SDS-PAGE and luciferase quantitated by luminometry assay (Fig. ). In unsupplemented reticulocyte lysates, the Luc-SL and Luc-MS2 reporters were translated at the same level, which was unaffected by the addition of MS2 protein (Fig. ). The addition of MS2-hSLBP activated translation to the same extent on both reporters, as previously described (24
). The addition of hSLIP1 had a small effect on the translation of the Luc-SL reporter and no effect on the Luc-MS2 reporter. In contrast, the addition of MS2-hSLIP1 had the same effect as SLIP1 on the Luc-SL reporter, and there was a much larger effect on the Luc-MS2 reporter, similar to that of MS2-SLBP. Thus, results suggest that the recruitment of SLIP1 to the 3′ end of the mRNA is sufficient to increase the translation of the Luc-MS2 reporter (Fig. ).
hSLIP1 activates the translation of histone mRNAs in mammalian cells.
To determine whether hSLIP1 also affected the translation of mRNAs ending in the histone stem-loop in mammalian cells, we utilized a GFP reporter gene ending in the histone stem-loop and quantified the expression of GFP by FACS analysis (31
). We compared the expression of the GFP-SL gene to that of the GFP-poly(A) gene (Fig. ). Transfection of hSLBP specifically stimulated expression of the stem-loop reporter as previously reported (28
). The transfection of hSLIP1 also stimulated the expression of the GFP-SL reporter but had a much smaller effect on the translation of the GFP-poly(A) reporter (Fig. ). The expression of both hSLIP1 and hSLBP together resulted in a similar increase in GFP expression as the expression of either protein alone.
FIG. 5. SLIP1 stimulates the translation of a histone stem-loop reporter in mammalian cells. (A) A reporter encoding GFP followed by the histone stem-loop and processing signals or a reporter encoding GFP followed by a polyadenylation signal (28, 31) was transfected (more ...)
The production of substantial amounts of GFP-SL mRNA from this reporter requires the expression of ZFP100, a U7 snRNP protein that is limiting for the expression of histone mRNA (28
). To further assess the role of SLIP1 in translation in mammalian cells, we transfected cells with both ZFP100 and the GFP-SL reporter. Twenty-four hours later, we transfected cells with increasing amounts of SLIP1 and then analyzed the levels of GFP-SL mRNA by Northern blotting and of GFP protein by use of FACS 48 h later. Increased levels of hSLIP1 increased the expression of the GFP protein up to fivefold in a concentration-dependent manner (Fig. ).
We determined whether the expression of hSLIP1 altered the amount of total GFP mRNA or processed GFP mRNA ending at the stem-loop. We measured the amount of GFP mRNA processed at the histone 3′ end in cells transfected with ZFP100 24 h before the transfection of hSLIP1 (28
) by use of S1 nuclease mapping (Fig. , lanes 1 to 4). There was no change in the amount of processed reporter mRNA or of readthrough reporter mRNA with transfection of increasing amounts of hSLIP1. We also performed Northern blot analysis for total GFP mRNA and 7SK RNA (as an internal control) on the same samples (Fig. , lanes 5 to 8). The expression of hSLIP1 stimulated GFP expression but did not increase the levels of GFP-SL mRNA in either of these assays, indicating that the increased GFP levels are due to increased translation of the GFP-SL mRNA. There was no change in the cell cycle distribution after the expression of SLIP1 or SLBP in these cells (not shown), indicating that these results are not due to an increase in the number of S-phase cells.
SLIP1 interacts with both SLBP and eIF4G.
To determine whether SLBP and SLIP1 interact in cells, we used the anti-hSLIP1 antibody to the C-terminal peptide, which efficiently precipitates SLIP1 from total cell lysates from HeLa cells (Fig. , top, lane 3). Western blotting of the immunoprecipitates for SLBP revealed that anti-hSLIP1 coprecipitates hSLBP (Fig. , middle, lane 3). The addition of RNase to the lysate prior to the immunoprecipitation only slightly reduced the amount of SLBP that was coimmunoprecipitated (Fig. , middle, lane 5). This demonstrates that endogenous SLBP and hSLIP1 interact in vivo and that the interaction is independent of RNA.
FIG. 6. SLIP1 interacts with SLBP and eIF4G in vitro and in vivo. (A) Lysates prepared from exponentially growing HeLa cells were immunoprecipitated with anti-myc (lanes 2 and 4) or anti-SLIP1 antibody against the C terminus of hSLIP1 (lanes 3 and 5). One-half (more ...)
Since PABP interacts with eIF4G to circularize mRNAs (8
), we also tested the SLIP1 immunoprecipitates for the presence of eIF4GI. eIF4GI was present in the immunoprecipitates (Fig. , bottom, lane 3). However, treatment of the lysate with RNase prior to immunoprecipitation resulted in the loss of eIF4GI from the immunoprecipitates (Fig. , bottom, lane 5).
Since the translation of polyadenylated mRNAs requires interaction between the 5′ end and the 3′ end of the mRNA mediated by PABP binding to eIF4G, we tested whether there was a direct interaction between SLIP1 and eIF4G. We synthesized eIF4GI and eIF4GII by in vitro translation and tested the ability of GST-hSLIP1 to bind to the 35S-labeled proteins (Fig. ). Both eIF4GI and eIF4GII bound to GST-hSLIP1 better than they bound to GST (Fig. , lane 3). We tested various fragments of eIF4GI and eI4GII for their abilities to interact with GST-hSLIP1. Fragments containing approximately the first 420 aa of eIF4GI and eIF4GII each interacted with GST-hSLIP1. The binding activity was present in aa 27 to 170 of eIF4GI (Fig. , middle, lane 6), while the fragment from 170 to 420 bound GST as well as it bound GST-hSLIP1 (Fig. , bottom, lanes 5 and 6). Similarly, the binding of hSLIP1 to eIF4GII aa 101 to 420 was weaker than the binding to aa 31 to 420, suggesting that hSLIP1 binds within aa 31 to 101.
To determine whether the interaction could occur between recombinant proteins, we tested the ability of His-tagged eIF4GI to interact with GST-hSLIP1 (Fig. ). GST-hSLIP1 bound to recombinant eIF4GI(27-129) but not to eIF4GI(123-420) (Fig. , lanes 3 and 6). Thus, hSLIP interacts with the N-terminal region of eIF4GI. The binding site for PABP is 172 to 200 in eIF4GI (8
). Thus, the binding site for hSLIP1 on eIF4GI is close to, but does not overlap with, the binding site for PABP. Additionally, the 27 to 129 fragment did not bind to recombinant SLBP (Fig. , lane 9), consistent with the inability of other investigators to detect a direct interaction between SLBP and eIF4G (7
SLIP1 does not bind directly to RNA (unpublished results). Note that SLIP1 was active both in frog oocytes and in mammalian cells using reporter mRNAs that lacked any histone sequences other than the stem-loop. Thus, the likely role of SLIP1 is to help circularize the mRNA by interacting with both SLBP and eIF4G and not directly with the mRNA.
We also examined the effect of HU on the association of SLIP1 with SLBP and eIF4G. The treatment of cells with HU results in a rapid reduction in histone mRNA levels, with no effect on SLBP levels (35
). We carried out immunoprecipitation experiments using cells stably expressing HA-hSLIP1. HA antibody was used to immunoprecipitate HA-tagged hSLIP1, and the presence of SLBP (Fig. ) or eIF4GI (Fig. ) was probed by Western blotting. Precipitation with an anti-myc antibody was used as a control. We detected SLBP associated with SLIP1, and this association was not affected by HU treatment (Fig. , lanes 3 and 6). However, the treatment of cells with HU resulted in the loss of SLIP1 interaction with eIF4GI (Fig. , lane 3), consistent with the loss of histone mRNA after HU treatment.
We interpret these experiments to imply that SLIP1 is bound to the translating histone mRNA and tethered to the 3′ end of the mRNA by SLBP and that it then interacts with eIF4GI at the 5′ end of the mRNA. The interaction between SLIP1 and SLBP is strong enough to be maintained in the absence of RNA. The interaction between SLIP1 and eIF4G is weaker, and the proteins do not remain associated during immunoprecipitation in the absence of RNA. These results are consistent with the results of Ling et al. that SLBP and eIF4G coimmunoprecipitate in an RNA-dependent manner (16
). We note that similar results were obtained with PABP, which also interacts with eIF4G. Coimmunoprecipitation of eIF4G with PABP from cells is also RNase sensitive, although the proteins clearly interact in vitro (8
SLIP1 is an essential protein in HeLa cells.
To establish whether SLIP1 is an essential gene, we used RNA interference (RNAi) to knock down hSLIP1 in HeLa cells (Fig. ). As a control, we also used RNAi to knock down SLBP, which slows progression through S phase without affecting viability (29
) (Fig. ). The treatment of HeLa cells with either siRNA1 or 2 against hSLIP1 resulted in a rapid depletion of hSLIP1 protein and cell death, with 50% of the cells dying within 24 h and over 75% dying by 72 h (Fig. ). HeLa cells treated with a control siRNA, C2 (29
), were not affected. Thus, unlike SLBP, hSLIP1 is essential for the viability of HeLa cells and other cell lines that we tested (data not shown). Each siRNA effectively depleted both HA-tagged hSLIP1 expressed from a transfected gene (Fig. , top, lanes 4 to 6) and the endogenous hSLIP1 (Fig. , middle, lanes 4 to 6), using the antibody to the full-length SLIP1 protein. A Western blot against the PTB protein was used as a loading control (Fig. , bottom).
FIG. 7. Knockdown of SLIP1 results in the death of HeLa cells. (A) Cells were transfected with two different siRNAs against SLIP1 (siRNA1, closed triangles; siRNA2, closed squares; siRNA 1 and 2 together, stars), a siRNA against SLBP (open diamonds), and a control (more ...)
To demonstrate that cell death was a result of knocking down hSLIP1, we created a gene expressing an RNAi-resistant form of hSLIP1 mRNA (mutating the siRNA1 target site). Expressing the RNAi-resistant form of hSLIP1 restored both cell growth (Fig. ) and expression of the SLIP1 protein (Fig. , lane 8) in cells treated with siRNA1 but not siRNA2, confirming that the cell death phenotype was due to SLIP1 depletion. There was no change in cell cycle distribution in the HeLa cells as a result of the knockdown of SLIP1 (Fig. ). Cell death was likely due to apoptosis, based on the loss of full-length caspase 9 (data not shown), and the accumulation of cells containing less than 2 N DNA content (data not shown). While the biological lesion in the cell that results in the activation of the apoptotic pathway is not known, knocking down SLBP, which has a larger effect on histone protein biosynthesis (Fig. ), does not result in cell death, suggesting that there may be another pathway for which SLIP1 is essential.
FIG. 8. Effect of SLIP1 and SLBP knockdown on histone protein synthesis. (A) HeLa cells were treated with siRNA for SLIP1 or SLBP for 4 days. Western blots (top, anti-SLIP1; middle, anti-SLBP; bottom, loading control) were done to assess the knockdown of each (more ...)
We also assessed the effect of SLIP1 depletion on endogenous histone mRNA levels and histone protein synthesis. For these experiments, we knocked down SLIP1 in HeLa cells for the minimal time that gave substantial knockdown (about 70%) (Fig. , top, lane 6) but retained cell viability. There was only a small decrease in SLBP protein levels in the SLIP1 knockdown cells (Fig. , middle, lane 6). We used the antibody against the C terminus of hSLIP1 for these analyses. We also knocked down SLBP in a parallel culture of cells and carried out the same analysis. The SLBP knockdown was about 75% effective (Fig. , middle, lane 7) and there was no change in SLIP1 levels in the SLBP knockdown cells (Fig. , top, lane 7). A cross-reacting band is shown as a loading control (Fig. , bottom).
We analyzed the levels of histone mRNA in these cells relative to the level of 7SK RNA, and there was not a significant change in histone mRNA levels in either SLBP knockdown or SLIP1 knockdown cells (Fig. ), consistent with there being no change in cell cycle distribution. A reduction of histone mRNA levels due to SLBP knockdown requires longer treatment with siRNA and a more complete knockdown of SLBP (E. J. Wagner and W. F. Marzluff, unpublished data).
To assess the rate of histone protein synthesis, we starved the siRNA-treated cells for methionine for 30 min, labeled the cells for 10 min with [35S]methionine, and resolved total nuclear proteins by SDS-PAGE. We also analyzed the cytoplasm of these same cells, and there was no newly synthesized histone protein in this fraction (not shown). We consistently saw higher levels of total methionine incorporation in the SLIP1 knockdown cells for unknown reasons and thus performed analyses loading either equal amounts of radioactive protein (Fig. , lanes 1 and 2) or equal amounts of total protein (Fig. , lanes 3 to 5). We quantified the amount of radiolabeled H2a/H3 histone using a PhosphorImager and compared the levels of the histone proteins to two prominent labeled bands of nonhistone protein (Fig. ). There was no change in the relative intensity of the nonhistone protein bands, as judged either by Coomassie staining (Fig. ) or by autoradiography (Fig. ). In the SLBP knockdown, there was a decrease of 50% in histone protein synthesis, consistent with a role for SLBP in translation. There was reproducibly a 20 to 30% decrease in histone protein synthesis when SLIP1 was knocked down. The ratio of the two control bands varied by only 5% among the various samples.
These experiments, together with the stimulation of the translation of the histone reporter mRNA (Fig. ), demonstrate a role for SLIP1 in the translation of mRNAs ending in the histone stem-loop in mammalian cells. The modest effect on translation in the SLBP and SLIP1 knockdown cells is likely due to the residual proteins present in the cells. Because SLIP1 depletion results in cell death while SLBP depletion does not, our data suggest that SLIP1 may function in other important cellular processes in addition to histone mRNA translation.