Since SLBP is the only known protein that specifically binds the 3′ end of histone mRNA, it is an obvious candidate to mediate the effect of the stem-loop on translation of histone mRNA. There are two SLBPs in frog oocytes, each of which binds the stem-loop with similar affinity (26
). Each of these proteins consists of about 280 amino acids, with the RBD located in the center of the protein.
Only xSLBP1 activates translation of Luc-SL mRNA in vitro.
To investigate the possible role of SLBP in translation, we used both an in vitro system, the RRL, and an in vivo system, injection of reporter mRNAs into Xenopus oocytes. We used three reporter luciferase mRNAs, which are shown in Fig. . The first (Luc-SL) ends in the histone stem-loop, the second (Luc-TL) ends in a GNRA tetraloop that contains the same stem and flanking sequences as the histone stem-loop but that does not bind SLBP, and the third (Luc-polyA) ends in a 50-nt poly(A) tail. We also used an mRNA encoding the Renilla luciferase that ended in a histone stem-loop (R-Luc-SL) as an internal control for the in vivo experiments. We determined the effect of expression SLBPs on the translation of the various reporter luciferase mRNAs in vitro and in vivo.
FIG. 1. SLBP stimulates histone mRNA translation in vitro. (A) Structure of the 3′ end of the luciferase reporter mRNAs. The Luc-SL mRNA ends in the histone stem-loop that binds SLBP, the Luc-TL mRNA ends in a stem-loop that does not bind SLBP, and the (more ...)
A schematic of the in vitro assay is shown in Fig. . Briefly, xSLBP1, xSLBP2, or any one of various chimeric or mutated proteins was first expressed in an RRL. This served as the source of SLBP for the in vitro assays. After incubation for 90 min, equal amounts of these lysates were added to a translation reaction mixture containing uncapped, polyadenylated CAT mRNA and a luciferase reporter mRNA, also uncapped. Thus, within each experiment, the different reporter mRNAs were tested with the same amount and preparation of SLBP.
The reticulocyte lysate contains very small amounts of SLBP (66
). Expression of SLBP in the lysate from synthetic mRNA results in the synthesis of sufficient SLBP to bind exogenous stem-loop RNA in a mobility shift assay (12
). Enough SLBP is produced by this approach to bind all of the reporter mRNAs added to the lysate. Since the reticulocyte lysate largely responds to the cap at the 5′ end of the mRNA, we used uncapped mRNAs in these experiments. There was no effect of any of the proteins (or different 3′ ends) when we used capped mRNAs expressed from the same constructs (data not shown). We included an uncapped polyadenylated CAT mRNA both to provide an internal control for translation efficiency and as a competitor for translation initiation factors to maximize the effect of the 3′ end of the luciferase mRNA (49
). We analyzed the expression of the xSLBPs, CAT, and luciferase proteins by gel electrophoresis, followed by autoradiography (Fig. ). We also assayed luciferase activity with a luminometer (Fig. ).
We first tested the effects of xSLBP1 and xSLBP2 on the translation of the Luc-SL, Luc-TL, and Luc-polyA mRNAs in vitro. When either buffer or in vitro-synthesized xSLBP2 was added to the reticulocyte lysate containing different reporter mRNAs, the Luc-SL and Luc-TL mRNAs were translated with similar efficiencies (Fig. , lanes 1 and 2 and 4 and 5, respectively). The Luc-polyA mRNA was translated about 20-fold more efficiently than the mRNAs ending in a stem-loop (Fig. , lane 3). In the presence of xSLBP2, the absolute amounts of translation of all of the mRNAs (including the CAT competitor) were reduced (Fig. , lanes 4 to 6) because of the presence of the additional competitor SLBP mRNA. The relative amounts of luciferase expression compared to CAT expression determined by PhosphorImager analysis was essentially the same for all three luciferase mRNAs (Fig. , lanes 1 to 3 versus lanes 4 to 6). In contrast, when xSLBP1 was included in the final reaction mixture, there was a four- to fivefold increase in the translation of the Luc-SL mRNA (Fig. , lane 8 versus lane 5), relative to the Luc-TL mRNA. There was no change in the translation of the Luc-polyA mRNA relative to that of the Luc-TL mRNA in the presence of xSLBP2 (Fig. , lanes 4 and 6) or xSLBP1 (Fig. , lanes 7 and 9).
To determine whether the effect of xSLBP1 was dependent on the interaction of xSLBP1 with the stem-loop, we replaced xSLBP1 with in vitro-synthesized hSLBP (the orthologue of xSLBP1) or two hSLBP mutant forms (hSLBPRR/KK
, Fig. ). Addition of hSLBP stimulated translation of the Luc-SL mRNA four- to fivefold, the same level as xSLBP1 (Fig. ). In the mutants, two conserved arginines at positions 10 and 11 in the RBD were replaced with lysines (hSLBPRR/KK
) or the tyrosines at positions 24 and 27 were replaced with two serines (hSLBPYY/SS
). Neither of these proteins binds the stem-loop with high affinity, as determined by mobility shift assay (12
). Both hSLBPRR/KK
failed to stimulate translation of the Luc-SL mRNA (Fig. ).
FIG. 2. SLBP must bind mRNA to stimulate translation. (A) Lysates containing hSLBP or two mutant hSLBPs that do not bind the stem-loop were incubated with the standard polyadenylated CAT mRNA and the Luc-TL or Luc-SL mRNA and assayed for luciferase activity. (more ...)
Competition experiments were also carried out to demonstrate that SLBP must bind the Luc-SL mRNA to stimulate translation. Short, uncapped RNA oligoribonucleotides containing either the stem-loop (SL) or the reverse stem (RS), which does not bind SLBP (45
), were added together with Luc-SL or Luc-TL mRNA to the translation reaction mixtures supplemented with xSLBP1. A 100-fold molar excess of the 30-mer SL RNA effectively inhibited the stimulation of translation of Luc-SL mRNA in the reaction mixtures containing xSLBP1 (Fig. ). Addition of a 200-fold molar excess of the RS oligoribonucleotide had no affect on the stimulation of translation of Luc-SL mRNA (Fig. ). Neither competitor oligoribonucleotide had any effect on the translation of the Luc-TL mRNA (Fig. ). We conclude that the stimulation of the translation of Luc-SL mRNA requires that it form a complex with xSLBP1.
xSLBP1 activates translation of Luc-SL mRNA in vivo.
We also tested whether xSLBP1 could stimulate translation in vivo by injecting similar reporter mRNAs into Xenopus
oocytes after overexpression of different SLBPs in vivo. In this case, the reporter mRNAs were capped (since uncapped mRNAs are very unstable in the oocytes) but they were otherwise identical to the three mRNAs used in the in vitro studies (Fig. ). To allow us to readily measure the relative translation of two different mRNAs in the same oocytes, we constructed a reporter mRNA that encoded the Renilla
luciferase (R-Luc-SL) in addition to the reporter mRNAs encoding firefly luciferase. We then independently assayed the Renilla
luciferase and firefly luciferase activities from the same samples, providing an internal control for the activity of the reporter mRNAs (39
Stage VI oocytes were injected (T0, Fig. ) in the cytoplasm with capped mRNA encoding an SLBP or with buffer. Synthesis of SLBP was allowed to proceed for 12 to 16 h prior to cytoplasmic injection of equimolar amounts of the different capped firefly luciferase reporter mRNAs and the R-Luc-SL mRNA at time T1. At time T2, 12 to 16 h after injection of the reporter mRNAs, the oocytes were harvested. The activities of the two luciferases were measured by luminometry, and the SLBP levels were measured by Western blotting and, in some cases, by mobility shift assay. For each experiment shown, the same batch of oocytes was used. There was variability in the magnitude of the activation observed among different batches of oocytes, but the qualitative effects of the SLBPs were identical.
FIG. 3. SLBP stimulates translation of Luc-SL mRNA in vivo. (A) Schematic of the in vivo assay. Xenopus oocytes were injected either with buffer or with a synthetic mRNA encoding an SLBP (T0). At 12 to 16 h later (T1), the oocytes were injected with the reporter (more ...)
To control for the stability of the mRNAs, we measured the level of luciferase mRNAs by S1 nuclease protection at the end of the incubation (see Fig. ). The ability of each of these reporter mRNAs (which were capped) to program luciferase activity in the reticulocyte lysate was also determined. All three reporter mRNAs had similar activities in the absence of competitor mRNAs or SLBPs, since the cap dominates the in vitro translation activity (data not shown) (14
FIG. 7. Effect of xSLBP1 deletion mutant forms on translation activation in vivo. (A) Oocytes were injected with xSLBP1 deletion mutant mRNAs (Fig. ) or buffer at T0. At T1, they were injected with the R-Luc-SL mRNA together with the Luc-SL (more ...)
We tested the effect of increased expression of xSLBP1 or xSLBP2 in vivo on the translation of the three luciferase reporter mRNAs. The xSLBP1 and xSLBP2 protein levels (measured by Western blotting) were each increased more than fivefold over endogenous levels after injection of the SLBP mRNA (Fig. ). The exogenous SLBPs persisted at the same levels at the end of the incubation, regardless of the nature of the luciferase transcripts injected at T1 (Fig. , lanes 3 and 4 and 7 and 8). We measured the activities of both the Renilla and firefly luciferases and normalized the firefly luciferase activity from each reporter mRNA by adjusting to a constant amount of Renilla luciferase activity. We then set the firefly luciferase activity observed with the Luc-TL mRNA at 1 and expressed the activity of the Luc-SL and Luc-polyA mRNAs relative to the activity of the Luc-TL mRNA.
There is some free xSLBP1 and xSLBP2 present in the cytoplasm of Xenopus
oocytes, as assayed by mobility shift assay, with a larger amount of free xSLBP1 (64
). Thus, when the reporter mRNAs are injected into oocytes without expression of exogenous SLBPs, the activity measured is affected by the presence of the endogenous SLBPs. Figure shows a time course of the relative expression of the reporter mRNAs following injection at T1
into the cytoplasm of oocytes that had previously been injected at T0
with buffer (top), xSLBP1 mRNA (middle), or xSLBP2 mRNA (bottom). In oocytes injected with buffer, translation of Luc-SL mRNA was two- to threefold greater than translation of Luc-TL mRNA (Fig. , top). The Luc-polyA mRNA was translated two to three times better than the Luc-SL mRNA and six- to eightfold better than the Luc-TL mRNA. This stimulation of translation of the Luc-SL mRNA is likely due to the endogenous xSLBP1 in stage VI oocytes, and the translation of the Luc-polyA mRNA is probably activated by embryonic PABP (63
). When xSLBP1 was overexpressed in the oocytes prior to injection of the reporter mRNAs, the translation of the Luc-SL mRNA was stimulated to a level similar to that observed with the Luc-polyA mRNA, increasing more than 15-fold at 16 h compared to the translation of the Luc-TL mRNA in this batch of oocytes (Fig. , middle). Note that what is shown in Fig. is relative luciferase activity. The absolute amount of activity of both luciferases increased at each time. In contrast, when xSLBP2 protein was overexpressed, the Luc-SL and Luc-TL mRNAs were translated to the same extent (Fig. , bottom), while translation of the Luc-polyA mRNA was not affected (data not shown). The reduction in translation of the Luc-SL mRNA likely reflects the ability of overexpressed xSLBP2 to compete with the endogenous pool of xSLBP1 for the injected Luc-SL mRNA.
To rule out the possibility that these results were due to different stabilities of the reporter mRNAs, we measured the levels of the reporter mRNAs with an S1 nuclease protection assay (see Fig. , lanes 1 and 2 and 13 and 14). These results demonstrate that xSLBP1 can activate translation of a capped reporter mRNA that ends in the histone stem-loop in frog oocytes. In the presence of overexpressed xSLBP1, the translational efficiency of Luc-SL is similar to the translational efficiency mediated by a poly(A) tail (Fig. , middle), suggesting that, in frog oocytes, xSLBP1 is as effective in activating translation as is PABP.
The N-terminal domain of xSLBP1 is required for activation of translation.
The two Xenopus
SLBPs can be arbitrarily divided into three domains: the RBD and the flanking N- and C-terminal domains (Fig. ). The Xenopus
SLBPs are only similar in the RBD, which is located in the center of each protein (64
). These domains were exchanged between the frog SLBPs to generate six chimeras that bind the histone stem-loop with similar affinities (26
) (unpublished results). To localize the region of xSLBP1 required for translation, we tested the abilities of these chimeric proteins to stimulate translation of the Luc-SL mRNA in vitro. A similar approach previously allowed us to determine the regions of xSLBP1 required for pre-mRNA processing (26
FIG. 4. Xenopus xSLBPs and the chimeric proteins. xSLBP1 and xSLBP2 were arbitrarily divided into three domains, the central 73-amino-acid RBD and the N- and C-terminal domains. These domains were interchanged to give the six chimeric proteins shown at the bottom. (more ...)
The various xSLBP chimeras were synthesized in the RRL, and the lysates containing these proteins was added to fresh lysate together with the CAT mRNA competitor and either Luc-TL or Luc-SL reporter mRNA as described in Fig. . As previously shown (Fig. ), xSLBP1 stimulated translation of the Luc-SL mRNA (Fig. , lanes 1 and 2) while xSLBP2 did not (Fig. , lanes 15 and 16). The three chimeras that contained the amino-terminal portion of xSLBP1 (1-1-2, 1-2-1, and 1-2-2) stimulated translation of the Luc-SL mRNA to similar extents (Fig. , lanes 3 to 8). Those chimeras containing the amino-terminal portion of xSLBP2 (2-1-1, 2-1-2, and 2-2-1) had no effect on the translation of the Luc-SL mRNA (Fig. , lanes 9 to 14). Similar results were obtained both by quantifying luciferase protein by PhosphorImager or luciferase activity by luminometry (Fig. , bottom). We conclude that the N-terminal region of xSLBP1 contains the region required for translational activation in vitro. Similar results were obtained when these chimeras were analyzed in vivo (data not shown).
FIG. 5. Assay of the effects of xSLBP chimeras on translation in vitro. Lysates containing the xSLBP chimeras shown in Fig. were incubated with the standard polyadenylated CAT mRNA and the Luc-TL or Luc-SL mRNA and assayed for luciferase activity. (more ...) Localization of the translation activation domain in the amino-terminal region of xSLBP1.
To demarcate the boundaries of the xSLBP1 translation activation domain and define a minimal protein required for activation of translation, we generated a number of deletions in the N- and C-terminal regions of xSLBP1 (Fig. ). We tested the ability of each deletion mutant to stimulate translation of the Luc-SL mRNA both in vitro and in vivo. A protein missing the first 68 amino acids (Δ68-1-1) was as effective as full-length xSLBP1 in stimulating translation in vitro (Fig. , lanes 1 to 4). Deletion of 13 additional amino acids (Δ81-1-1) resulted in a loss of translation activation (Fig. , lanes 7 and 8). Deletion of the entire C-terminal portion of xSLBP1 (1-1-0 and Δ68-1-0) had no effect on activation of translation (Fig. , compare lanes 3 and 4 with lanes 5 and 6, lanes 7 and 8 with lanes 9 and 10, and lanes 1 and 2 with lanes 11 and 12).
FIG. 6. Effect of xSLBP1 deletion mutant forms on translation activation in vitro. (A) Deletions in the amino- and carboxy-terminal regions of xSLBP1. (B) Lysates containing the xSLBP1 deletion mutant forms shown in panel A were incubated with the standard polyadenylated (more ...)
We tested the same deletion mutants in vivo with essentially similar results (Fig. ). Mutant forms 1-1-0, Δ68-1-1, and Δ68-1-0 stimulated translation in vivo to extents similar to that of xSLBP1, whereas the Δ81-1-1 deletion was inactive. We assayed for the presence of these proteins in Xenopus oocytes by measuring RNA binding activity (Fig. ) and, when possible, by Western blotting (Fig. ). Mutant forms Δ68-1-0 and 1-1-0 are not detectable by Western blotting because the antibody against xSLBP1 recognizes only the C-terminal portion of the protein. The Δ81-1-1 mutant form was clearly overexpressed (Fig. , lanes 9 and 10, and C, lanes 7 and 8;) in the oocytes. The amounts of the Δ68-1-1 and Δ68-1-0 proteins active in stem-loop binding were greater than the store of endogenous xSLBPs (Fig. , lanes 5 to 8 versus lane 15), although they were overexpressed to lesser extents than the other proteins. The 131-amino-acid mutant form Δ68-1-0 represents the smallest xSLBP1 protein we tested that is capable of activating translation of a reporter gene ending in the stem-loop.
The data provided by the deletion mutants suggested that the critical region for translation activation is located between amino acids 69 and 81. To confirm the importance of these residues, we deleted amino acids 69 to 81 from the full-length protein (Fig. , Δ13). The Δ13 mutant did not stimulate translation of the Luc-SL mRNA in vitro (Fig. , lanes 13 and 14) or in vivo (Fig. ). The Δ13 protein was clearly overexpressed in the oocytes, as shown by both mobility shift assay (Fig. , lanes 11 and 12) and Western blotting (Fig. , lanes 5 and 6). Note that the deletion of 13 amino acids caused a major change in the mobility of the protein on SDS-polyacrylamide gels both after synthesis in vitro (Fig. , lanes 13 and 14) and after synthesis in vivo (Fig. , lanes 5 and 6). xSLBP1 has aberrant mobility on SDS-PAGE (apparent molecular mass of 45 kDa for a 31-kDa protein) (64
). Deletion of these 13 amino acids resulted in mobility similar to that expected for the 30-kDa Δ13 protein.
The amount of the Luc-SL and Luc-TL mRNAs present in the oocytes after the incubation was determined by an S1 nuclease protection assay (Fig. ). A probe labeled in the coding region of the Luc-SL gene is protected by the Luc-SL mRNA to the 3′ end of the mRNA and by the Luc-TL mRNA to the place in the 3′ UTR where the Luc-TL sequence differs from the Luc-SL sequence. Since there is a single labeled nucleotide in each probe, the intensity of the protected fragment is proportional to the amount of RNA present in the sample. The amounts of both mRNAs were similar in the oocytes injected with each of the mutant SLBPs, demonstrating that the differences in translation efficiency were not due to variations in reporter mRNA stability.
Amino acids 70 to 84 are essential for translation.
To precisely define the amino acids in xSLBP1 required for translation activation, we generated alanine scanning mutations in each of which a stretch of five amino acids was replaced with alanines spanning residues 65 to 89 (Fig. ), leaving the rest of xSLBP1 intact. The effects of these mutant forms on translation activation were analyzed with both in vitro (Fig. ) and in vivo (Fig. ) assays. All of the mutant forms were expressed to similar extents in vitro (Fig. ) and in vivo (Fig. ). Note that one of the mutant proteins, SLBP65-69/5A, when expressed both in vitro (Fig. , lanes 5 and 6) and in vivo (Fig. , lanes 3 and 4), has slower mobility than full-length SLBP. The xSLBP65-69/5A and xSLBP85-89/5A mutant proteins, which have mutations outside the region deleted in the Δ13 mutant protein, had translation activity similar to that of wild-type xSLBP1 in vitro (Fig. , lanes 5 and 6 and 13 and 14) and in vivo (Fig. ). Luciferase synthesis was increased six- to sevenfold in these experiments from the Luc-SL mRNA compared to the Luc-TL mRNA. The xSLBP70-74/5A and xSLBP75-79/5A mutant proteins each were inactive in translation in vitro (Fig. , lanes 7 to 10) and in vivo (Fig. ). Expression of xSLBP70-74/5A and xSLBP75-79/5A in oocytes reduced translation of the Luc-SL mRNA, consistent with these mutant proteins competing with the endogenous xSLBP1 protein for binding of the Luc-SL mRNA but being unable to activate translation. The xSLBP80-84/5A mutant protein has about 50% of the activity of wild-type xSLBP1 in activation of translation in vitro (Fig. , lanes 11 and 12) and in vivo (Fig. ). Thus, the 10-amino-acid region of xSLBP1 between amino acids 70 and 80 is essential for the activation of translation of Luc-SL mRNA in both assays.
FIG. 8. Alanine scanning of the xSLBP1 translation activation domain. (A) Amino acids 65 to 89 in the N-terminal region of xSLBP1 were replaced sequentially, five residues at a time, with five alanines (underlined). WT, wild type. (B) Lysates containing the five-alanine (more ...) The MS2-SLBP fusion protein activates translation of Luc-MS2 mRNA.
To determine whether SLBP has to be bound to the histone stem-loop or simply physically associated with the mRNA to activate translation, we constructed a fusion protein that had the entire hSLBP fused to the MS2 protein (MS2-hSLBP). We also constructed a luciferase reporter gene that had a binding site for the MS2 protein at the 3′ end of the mRNA. We expressed the MS2-hSLBP, the hSLBP, and the MS2 protein in the reticulocyte lysate and tested each for the ability to activate translation of the Luc-MS2, Luc-SL, or Luc-TL mRNA (Fig. ). The MS2-hSLBP fusion protein activated translation of both the Luc-MS2 and Luc-SL mRNAs 1.5- to 2-fold (range of three independent experiments), whereas it had no effect on the translation efficiency of the Luc-TL mRNA. The fusion of the MS2 protein to the hSLBP reduced its activity on the Luc-SL mRNA, and the fusion protein was equally active on the Luc-SL and Luc-MS2 mRNAs. Not surprisingly, in vitro-synthesized hSLBP failed to affect translation of the Luc-TL and Luc-MS2 mRNAs, although it activated Luc-SL mRNA translation nearly threefold. Control protein MS2 failed to activate translation of any of the reporter mRNAs tested (Fig. , lanes 1 to 3). Thus, the MS2-hSLBP can activate translation even though the SLBP is not directly bound to the mRNA. The reduced activity of this hybrid protein compared with that of the hSLBP, observed both on the Luc-SL and Luc-MS2 mRNAs, is likely due to the reduced activity of the hybrid protein in activating translation. Most likely, this is a nonspecific effect of the fusion of the MS2 protein to the SLBP that may well cause steric problems in interactions with the translation initiation machinery. We have no evidence that the SLBP RBD-stem-loop complex plays a direct role in translation. When we expressed the SLBP-MS2 fusion in oocytes, we also saw a lower, but equivalent, activity of this protein in activation of translation of the capped Luc-MS2 and Luc-SL transcripts (data not shown). Thus, the translation activation region of SLBP must be localized to the mRNA to mediate activation of translation (Fig. and , lanes 7 and 9 versus lane 8).
FIG. 9. Translation activation by MS2-hSLBP fusion protein in vitro. Lysates containing MS2 (lanes 1 to 3), MS2-hSLBP fusion protein (lanes 4 to 6), or hSLBP (lanes 7 to 9) were incubated with the standard polyadenylated CAT mRNA and the Luc-TL, Luc-SL, or Luc-MS2 (more ...)