Coupled tRNASec-Dependent Assembly of the Selenocysteine Decoding Apparatus

Molecular Cell, Vol. 11, 773–781, March, 2003, Copyright 2003 by Cell Press

Coupled tRNASec-Dependent Assembly of the Selenocysteine Decoding Apparatus Ann Marie Zavacki,1,3 John B. Mansell,1,3,4 Mirra Chung,1 Boris Klimovitsky,1,2 John W. Harney,1 and Marla J. Berry1,2,* 1 Thyroid Division Department of Medicine Brigham and Women’s Hospital and Harvard Medical School Boston, Massachusetts 02115 2 Department of Cell and Molecular Biology University of Hawaii at Manoa Honolulu, Hawaii 96822

Summary SECIS elements recode UGA codons from “stop” to “sense.” These RNA secondary structures, present in eukaryotic selenoprotein mRNA 3ⴕ untranslated regions, recruit a SECIS binding protein, which recruits a selenocysteine-specific elongation factor-tRNA complex. Elucidation of the assembly of this multicomponent complex is crucial to understanding the mechanism of selenocysteine incorporation. Coprecipitation studies identified the C-terminal 64 amino acids of the elongation factor as sufficient for interaction with the SECIS binding protein. Selenocysteyl-tRNA is required for this interaction; the two factors do not coprecipitate in its absence. Finally, through promoting this interaction, selenocysteyl-tRNA stabilizes the C-terminal domain of the elongation factor. We suggest that the coupling effect is critical to preventing nonproductive decoding attempts and hence forms a basis for effective selenoprotein synthesis. Introduction Translational fidelity is essential to achieving the correct readout of the genetic code. The deleterious consequences of misincorporating amino acids, premature termination, or inappropriate suppression of nonsense codons are apparent from the large number of heritable genetic disorders resulting from missense or nonsense mutations. Selenocysteine incorporation is a unique exception in decoding, presenting a challenge to fidelity, since selenocysteine is encoded by UGA, which also serves as a termination codon. The translational apparatus must therefore distinguish which function to perform at each UGA codon in the cell. Inefficient incorporation of selenocysteine into selenoproteins would result in premature termination and nonfunctional truncated products. Conversely, inappropriate incorporation of the amino acid at UGA stop codons would extend proteins beyond their encoded length, possibly leading to misfolded C termini. *Correspondence: [email protected] 3 These authors contributed equally to this work. 4 Present address: A.J. Park, Intellectual Property Lawyers and Consultants, Wellington, New Zealand.

Specific secondary structures in selenoprotein mRNAs, termed SECIS elements, are responsible for the fidelity of decoding UGA as selenocysteine. Their absence in other mRNAs results in the default termination function for UGA. In prokaryotes, the SECIS elements are located just downstream of UGA selenocysteine codons. Prokaryotic SECIS elements recruit a specialized factor, SELB, consisting of an elongation factor domain specific for selenocysteyl-tRNA (Sec-tRNA[Ser]Sec), and a SECIS RNA binding domain. The functions of Sec-tRNA[Ser]Sec recognition and delivery to the ribosome for incorporation into the nascent protein, dissociation of the empty elongation factor from the ribosome and from the SECIS element, and reassembly of the Sec-tRNA[Ser]Sec-elongation factor-SECIS complex for a subsequent round of incorporation, all reside within this single factor (Baron et al., 1993; Atkins et al., 1999). Eukaryotes, in contrast, employ two distinct factors for these processes. The first, termed SECIS binding protein 2 (SBP2) (Copeland et al., 2000), binds the SECIS element in the 3⬘ untranslated region (3⬘UTR) of the mRNA and recruits the second protein, the Sec-tRNA[Ser]Sec-specific elongation factor, EFsec (Tujebajeva et al., 2000; Fagegaltier et al., 2000). In previous studies, we showed that EFsec and SBP2 could be coimmunoprecipitated following coexpression in transfected cells (Tujebajeva et al., 2000). Thus, instead of the single, bifunctional protein found in prokaryotes, eukaryotes employ two distinct but physically interacting proteins for SECIS recognition and Sec-tRNA[Ser]Sec delivery. The physical separation of the SECIS binding and tRNA[Ser]Sec binding/delivery activities in eukaryotes, as well as the difference in locations of the SECIS elements (3⬘UTR versus coding region in prokaryotes), led to speculation that mechanistic differences would exist in the selenocysteine incorporation process in these two kingdoms (Berry et al., 1993; Tujebajeva et al., 2000; Atkins et al., 1999). In prokaryotes, SELB must dissociate from the SECIS element after delivery of the tRNA[Ser]Sec, as the ribosome must melt the SECIS stem-loop and translate through it. In eukaryotes, because the SECIS element is in the 3⬘UTR, there is no need for SBP2 to dissociate from it. Instead, one can envision at least two possible mechanistic scenarios. In the first, EFsec, SBP2, and the SECIS element could form a complex that remains associated on the mRNA, with EFsec delivering Sec-tRNA[Ser]Sec to the ribosome, then binding a new SectRNA[Ser]Sec for the next round of incorporation. In this case, once EFsec and SBP2 formed a complex, they would not dissociate. In the second scenario, the SBP2SECIS complex could remain intact, but EFsec would dissociate from SBP2 upon delivery of Sec-tRNA[Ser]Sec to the ribosome. The SBP2-SECIS complex would then recruit a new Sec-tRNA[Ser]Sec-EFsec complex for each round of selenocysteine incorporation. In the first case, it would be unlikely that tRNA[Ser]Sec would affect the interaction between EFsec and SBP2, whereas the second scenario would predict that the presence or absence of tRNA[Ser]Sec would influence the interaction between the two protein factors.

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SBP2. Coimmunoprecipitation of SBP2 was easily seen with the C-terminal region of EFsec (Figure 1, lane 8). No coprecipitation was detected with the N-terminal half (1–305; Figure 1, lane 7) or three-fourths (1–448; Figure 1, lane 6) of EFsec. Thus, the SBP2 interaction region is localized to the C-terminal domain of EFsec, and the in vitro expressed C-terminal fragment does not require other cellular factors to interact with SBP2. This domain is capable of interacting with SBP2 on its own. Figure 1. The C-Terminal Domain of EFsec Coimmunoprecipitates In Vitro with SBP2, but the Full-Length Protein Does Not Constructs expressing the indicated amino acids from EFsec were transcribed and translated in vitro in rabbit reticulocyte lysates. In vitro translated c-myc-tagged SBP2 was added to reactions, and coimmunoprecipitations were performed with anti-c-myc-agarose, followed by SDS-PAGE and autoradiography. Lanes 1–4, in vitro translation reactions; lanes 5–8, coimmunoprecipitations.

Herein, we present an emerging picture of the molecular interactions between EFsec and SBP2. This includes delineation of the specific regions in EFsec responsible for interaction, and demonstration of the requirement for the EFsec ligand, Sec-tRNA[Ser]Sec, for EFsec-SBP2 binding. Our findings suggest that Sec-tRNA[Ser]Sec, by orchestrating complex formation and dissociation, mediates EFsec-SBP2 delivery of the Sec-tRNA[Ser]Sec cargo to its destination, a UGA codon in a selenoprotein mRNA occupying the ribosomal A site. Results Mapping of the EFsec-SBP2 Interaction Domain We previously showed that two factors mediating cotranslational selenocysteine incorporation, EFsec and SBP2, could be coimmunoprecipitated following expression of both proteins by transient transfection in mammalian cells (Tujebajeva et al., 2000). We interpreted this result to indicate either a direct interaction between the two proteins or the presence of both proteins in a complex, possibly through bridging interactions with other factors in the cell. To assess whether the interaction was direct, i.e., occurred in vitro in the absence of other factors, we performed coimmunoprecipitation studies of the proteins following in vitro translation in rabbit reticulocyte lysates in the presence of 35S-methionine. The c-myc tag was introduced at the N terminus of SBP2 and the HA tag at the N terminus of EFsec. When antisera against c-myc were used for immunoprecipitation, followed by SDS-PAGE and autoradiography to detect labeled EFsec protein, no coprecipitation of EFsec could be detected (Figure 1, lane 5). Similarly, no coprecipitation of SBP2 could be detected when HAtagged EFsec was used as the bait (data not shown). This suggested the possibility that other cellular factors that are missing from the reticulocyte lysate might be required for the interaction of these two proteins. As the N-terminal region of EFsec was known to exhibit homology with EF1A, and the C-terminal region constitutes an extension relative to the standard elongation factor, we next assessed whether either of these domains when expressed alone could interact with

Deletion Mapping and Mutagenesis of the SBP2 Interaction Domain Further deletion mapping of the C-terminal extension was used to more precisely delineate the SBP2 interaction region, using exon-exon junctions as guidelines. The constructs used are shown schematically in Figure 2. In vitro translation of EFsec deletion constructs and c-myc-tagged SBP2, followed by immunoprecipitation of SBP2 using anti-c-myc antibodies, resulted in coprecipitation of EFsec fragments consisting of amino acids 448–583, 448–520, and 469–583 (Figure 3A). Use of bacterially expressed GST-tagged SBP2 and glutathione agarose to pull down the EFsec fragments resulted in an indistinguishable pattern of coprecipitation (Figure 3B). This further argues against a requirement for other cellular factors in the EFsec-SBP2 interaction, as the bacterial and mammalian selenocysteine incorporation machinery are not compatible. When the experiment was performed in reverse, using GST-fusions of the EFsec domains as the bait to coprecipitate SBP2, the efficiency was greatly reduced, but interactions were seen with fragments consisting of amino acids 448–583, 469–583, and 520–583 (Figure 3C, lanes 2, 3, and 6). The latter fragment was not detected in the in vitro translation experiments, presumably due to inefficient expression or rapid turnover of this small peptide. In this case, the interactions were strongest with the most C-terminal fragments, 469–583 and 520– 583. The decreased interaction with GST-EFsec as bait, relative to that with c-myc-SBP2 or GST-SBP2, may be due to effects of the large GST fusion on conformation or accessibility of the EFsec domains. Nonetheless, these results suggest that multiple domains in the C-terminal region of EFsec may interact with SBP2, but implicate the most C-terminal region as having the strongest interaction. Analysis of the entire protein sequence using ExPASy software (http://www.us.expasy.org) predicts the region from 520 to 583 to be highly surface accessible. Due to the strong GST pull-down reaction and the surface accessibility prediction of the 520–583 region, we focused predominantly on this region for more detailed analysis. Mutational Analysis of the SBP2 Interaction Domain in EFsec To further assess regions potentially involved in the EFsec-SBP2 interaction, we introduced single or multiple point mutations at amino acids that are conserved among the known eukaryotic EFsec sequences. The sequences in this region, conserved amino acids, and positions of mutations are shown in Figure 4A, and the specific substitutions are given in Table 1. The effects of these mutations on the interactions between EFsec

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Figure 2. EFsec Constructs Used for Interaction Domain Mapping Constructs expressing the indicated regions of EFsec were subcloned into pGBK with N-terminal HA epitope tags. Black lines indicate exon-exon boundaries.

448–583 and SBP2 were assessed by GST pull-down, and the amount of GST-EFsec 448–583 mutant in each pull-down reaction was normalized to the amount of expressed protein added, as determined by Western analysis. Mutations introduced in the 520–583 region resulted in ⵑ30%–60% decreases in coprecipitation between EFsec and SBP2 (Figure 4B), compared to the interaction achieved with the corresponding wild-type EFsec domain. The exception in this region was R538E, which had no effect. In contrast, none of the mutations in the 448–520 region inhibited coprecipitation (data not

shown), pointing to the 520–583 region as being the most critical for the EFsec-SBP2 interaction. Role of Sec-tRNASec in Complex Formation and EFsec Protein Stability Our ability to demonstrate in vitro interaction of SBP2 with the C-terminal domain of EFsec, but not full-length EFsec, suggested the possibility that the presence of the N-terminal region might be inhibitory to the interaction. Further, the contrast between the in vitro results and our previous in vivo result, showing coprecipitation of

Figure 3. Discrete Regions in the C-Terminal Domain of EFsec Interact with SBP2 Constructs expressing the indicated amino acids from EFsec were transcribed and translated in vitro in rabbit reticulocyte lysates or were expressed as GST-fusions in E. coli. C-myc-SBP2 was translated in vitro, or GSTSBP2 was expressed in E. coli, as described in Experimental Procedures. Coprecipitations were performed with anti-c-myc-agarose (A) or glutathione-agarose (B and C), followed by SDS-PAGE and autoradiography. Left panels, in vitro translation reactions; right panels, coprecipitations or GST pull-downs.

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Figure 4. Role of EFsec C-Terminal Domain Conserved Amino Acids in EFsec-SBP2 Interaction (A) Alignment of EFsec C-terminal domain sequences. Conserved amino acids are highlighted in gray, and mutated amino acids are indicated by asterisks. (B) GST pull-downs of EFsec C-terminal domain mutants. GST-fusion constructs expressing the EFsec 448–583 C-terminal domain with the indicated mutations were expressed in E. coli, and crude extracts were incubated with in vitro translated SBP2, followed by pulldown with glutathione-agarose, SDS-PAGE, and autoradiography (upper panel). Aliquots of bacterially expressed EFsec mutants were analyzed by Western blotting with an antiGST antibody to verify expression levels (lower panel).

the full-length proteins, suggests that factors that are present in vivo but not in vitro might play a role in this interaction. A likely candidate for such a factor is SectRNA[Ser]Sec, the ligand for the N-terminal elongation factor domain of EFsec. We tested the effects of cotransfection of the tRNA[Ser]Sec gene on the interaction between EFsec and SBP2 in transfected cells. Strikingly, the amount of coprecipitation in the presence of the cotransfected

tRNA[Ser]Sec gene was dramatically enhanced over that seen in its absence (Figure 5A, upper panel, lanes 7 and 8 versus 5 and 6). We next examined whether the effect of tRNA[Ser]Sec on EFsec-SBP2 interaction was limited solely to full-length EFsec, or whether similar effects could be seen with a truncated EFsec protein. tRNA[Ser]Sec cotransfection experiments were thus performed with SBP2 and the C-terminal domain of EFsec (amino acids

Table 1. Point Mutations in the C-Terminal Domain of EFsec Mutation

Effect on SBP2 Interaction

Species Conservation

K 460 A F 481 A K 482 A S 498 A I 505 A D 506 A I 530 E K 536 E K 537 E R 538 E V 562 D L 566 D R 570 E Y 571 E

None None None None None None Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased

M. M. M. M. M. M. M. M. M. M. M. M. M. M.

musculus, musculus, musculus, musculus, musculus, musculus, musculus, musculus, musculus, musculus, musculus, musculus, musculus, musculus,

H. H. H. H. H. H. H. H. H. H. H. H. H. H.

sapiens, sapiens, sapiens, sapiens, sapiens, sapiens sapiens sapiens, sapiens, sapiens sapiens sapiens sapiens sapiens

C. C. C. C. C.

elegans elegans elegans elegans elegans, M. jannaschi

C. elegans, M. jannaschi C. elegans

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Figure 5. Effect of tRNASec and SBP2 Expression on EFsec-SBP2 Coimmunoprecipitation and EFsec Protein Levels In Vivo (A) EFsec full-length protein (full) or 448–583 C-terminal domain (C term) with N-terminal FLAG epitope tags was coexpressed in transfected HEK-293 cells with SBP2 in the presence or absence of the tRNASec gene. Cell lysates were subjected to immunoprecipitation with anti-SBP2 antibody (upper panel). Aliquots were analyzed by Western blotting with anti-FLAG antibody (lower panel). (B) FLAG-tagged EFsec full-length protein or 448–583 C-terminal domain was expressed as above in the presence or absence of SBP2 and tRNASec expression plasmids, as indicated. Cell lysates were analyzed by Western blotting with anti-FLAG antibody. (C and D) 35S-methionine in vivo labeling was carried out following expression of the indicated plasmids in transfected cells. After 1 hr of labeling, media were changed and unlabeled methionine was added. Incorporation of unlabeled methionine and decay of labeled proteins was allowed to proceed for the indicated times, followed by immunoprecipitation with anti-FLAG antibody, SDS-PAGE, and autoradiography.

386–583). Quite surprisingly, coexpression of tRNA[Ser]Sec resulted in a striking enhancement in coprecipitation of the C-terminal EFsec fragment with SBP2 (Figure 5A, upper panel, lanes 11 and 12 versus 9 and 10). Thus, the C-terminal region of EFsec, and not just the N-terminal elongation factor domain, apparently interacts with tRNA[Ser]Sec. Western analysis indicates that for the full-length protein, the increase in coimmunoprecipitation could not be attributed solely to changes in EFsec protein level, as the amount of full-length EFsec was only slightly increased in the presence versus the absence of coexpressed tRNA[Ser]Sec (Figure 5A, lower panel, lanes 7 and 8 versus 5 and 6). In contrast, the amount of EFsec C-terminal fragment was increased in the presence of coexpressed tRNA[Ser]Sec to nearly the same extent that coprecipitation was enhanced (Figure 5A, upper and lower panels, lanes 11 and 12 versus 9 and 10), suggesting that tRNA[Ser]Sec affects stability of the truncated protein, rather than interaction with SBP2. Effect of SBP2 on EFsec Protein Stability We next assessed the effects of SBP2 on EFsec protein levels in transfected cells. Coexpression of SBP2 was

found to increase the amount of full-length EFsec protein by ⵑ1.6-fold in the absence of coexpressed tRNA[Ser]Sec, suggesting a stabilizing effect of complex formation on EFsec (Figure 5B, lane 3 versus 9). This effect was slightly greater in the presence of the tRNA (lane 4 versus 10). Stabilization of the C-terminal domain of EFsec by SBP2 coexpression was even more pronounced, suggesting that the truncated protein is rapidly turned over in the absence of SBP2. Quantitation of protein levels indicated that cotransfection of SBP2 increased the levels of EFsec 386–583 protein by 3-fold in the absence and 6-fold in the presence of the cotransfected tRNA gene (Figure 5B, lanes 5 and 6 versus 11 and 12). Finally, in the absence of cotransfected SBP2, the stabilizing effect of tRNA[Ser]Sec coexpression on truncated EFsec protein was abolished (Figure 5B, lane 11 versus 12). Thus, the stabilizing effect of the tRNA is dependent on the presence of SBP2 and presumably occurs through promoting the protein-protein interaction. Coexpression of the tRNA[Ser]Sec gene in the presence of SBP2 resulted in a further ⵑ1.9-fold increase in levels of truncated EFsec 386–583 (Figure 5B, lane 6 versus 5) beyond the 3-fold increase seen with SBP2 alone. To investigate the reason for the increases in EFsec 386–583 protein levels seen with SBP2 and tRNA[Ser]Sec

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presumably by competing with the ability of full-length EFsec to interact. Discussion

Figure 6. The C-Terminal Domain of EFsec Competes for Selenocysteine Incorporation In Vivo EFsec 448–583 C-terminal fragment was expressed in transfected HEK-293 cells with a type 1 deiodinase selenoenzyme expression plasmid or a cysteine mutant type 1 deiodinase. Type 1 deiodinase enzyme activity was assayed in cell lysates as described previously.

coexpression, we performed pulse-chase labeling experiments to assess protein turnover rates. Cells were transfected with EFsec expression plasmids in the presence or absence of SBP2 and tRNA[Ser]Sec expression plasmids. Two days later, 35S-methionine labeling was carried out for 1 hr, followed by chasing with unlabeled methionine for varying periods of time. Lysates were harvested and immunoprecipitations carried out with anti-FLAG antibody. This showed increases in the total amount of both full-length EFsec protein (Figure 5C) and the truncated protein (Figure 5D) in the presence of SBP2 and tRNA[Ser]Sec versus the levels seen in the absence of the added factors. The half-life of the full-length protein was greater than 12 hr, with the effect of the factors on EFsec protein levels ranging from 1.3-fold to greater than 2-fold. The half-life of EFsec 386–583 was dramatically reduced compared to the full-length protein, to between 30 and 60 min. Coexpression of SBP2 and tRNA[Ser]Sec significantly stabilized the truncated protein, increasing the half-life to between 60 and 120 min and increasing the level of truncated protein by 2- to 3-fold. Thus, the interaction between the C-terminal domain of EFsec, tRNA[Ser]Sec, and SBP2 results in stabilization of the elongation factor C-terminal domain. Expression of the EFsec C-Terminal Domain Competes for Selenocysteine Incorporation To determine whether the EFsec C-terminal domain, via its interactions with SBP2 and tRNA[Ser]Sec, might inhibit selenocysteine incorporation by sequestering the protein factor and/or ligand, we investigated the effect of overexpressing the C-terminal domain of EFsec on selenocysteine incorporation. When this expression plasmid was cotransfected with a wild-type, type 1 deiodinase expression plasmid, a dose-dependent inhibition of deiodinase activity was seen. In contrast, the EFsec plasmid had no effect on enzyme activity expressed from a cysteine mutant deiodinase expression plasmid (Figure 6), indicating that the competition effect was specific for selenocysteine incorporation. Thus, the C-terminal domain of EFsec is capable of inhibiting SBP2 function,

The existence of proteins with multiple functional domains allows the physical linkage of disparate functions. Conversely, separation of functions into distinct but physically interacting proteins provides greater opportunity for regulatory fine tuning. The identification of two separate eukaryotic proteins serving analogous functions to the single bacterial selenocysteine-specific elongation factor, SELB, raises intriguing questions about the evolution and regulation of selenoprotein synthesis in eukaryotes. Efficient selenocysteine incorporation is necessary not only to avoid the energy expenditure of generating nonfunctional truncated peptides. In addition, decoding UGA as selenocysteine rather than stop is required to circumvent degradation of selenoprotein mRNAs via the nonsense decay pathway (Moriarty et al., 1998; Weiss and Sunde, 1998). Further, the fact that a single SECIS element can and does serve in decoding multiple UGA codons suggests that the mechanism of incorporation in eukaryotes has evolved to allow rapid recycling and reassembly of selenocysteine incorporation complexes. This is particularly crucial in view of the fact that tRNA[Ser]Sec and SBP2 are limiting in most cells and tissues (Berry et al., 1994; Low et al., 2000; Copeland et al., 2000). To further our understanding of the mechanisms of assembly and function of the eukaryotic selenocysteine incorporation machinery, we investigated the interactions between the two protein factors, EFsec and SBP2, and the influence of Sec-tRNA[Ser]Sec on this interaction. Based on our previous demonstration of coimmunoprecipitation of the two proteins from transfected cell extracts (Tujebajeva et al., 2000), we sought to delineate the specific region of EFsec responsible for this interaction. Surprisingly, this led to the observation that the two full-length proteins did not appear to interact in vitro in the absence of other cellular factors. Thus, we investigated the role of the EFsec RNA ligand, tRNA[Ser]Sec, in the interaction between the two proteins. A precedent for such a role had been established from studies of selenoprotein synthesis in E. coli. Early studies showed that SELB forms a tighter complex with the SECIS element in the presence of Sec-tRNASec than in its absence (Baron et al., 1993). More recently, stopped-flow kinetic analysis was used to demonstrate that the presence of SectRNA[Ser]Sec significantly slows the rate of dissociation of SELB from a bacterial SECIS RNA minihelix (Thanbichler et al., 2000). In the present study, we show that Sec-tRNA[Ser]Sec is required for EFsec-SBP2 interaction. We suggest that this occurs through conformational changes affecting RNA-protein (Sec-tRNA[Ser]Sec-EFsec) and protein-protein (EFsec-SBP2) interactions. This is depicted in the model in Figure 7, in which binding of Sec-tRNA[Ser]Sec by EFsec triggers a conformational change, indicated by bending of the C-terminal domain upward (Figure 7A). This change in EFsec allows interaction with SBP2 such that the complex can then deliver Sec-tRNA[Ser]Sec to the UGA codon at the ribosome. Upon delivery and release of

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Figure 7. Model for the Effects of tRNA Binding on EFsec Conformation, EFsec-SBP2 Interaction, and Ribosome Interaction (A) Binding of tRNASec by EFsec triggers a conformational change, indicated by bending of the C-terminal domain upward, allowing interaction with SBP2. SBP2 can then deliver tRNASec to the UGA codon at the ribosome. (B) Delivery of Sec-tRNA at the ribosome triggers a reversion of EFsec to the pre-tRNA binding conformation, which disfavors SBP2 interaction, releasing EFsec to bind a new Sec-tRNASec molecule and allowing SBP2 to recruit a new Sec-tRNASec-EFsec complex. The mRNA, including the SECIS element in the 3⬘UTR, is indicated by the thin black line. The open reading frame is indicated by the thick black line, with double gray ovals representing ribosomes on the mRNA. SBP2 and EFsec are labeled, with the C-terminal domain of EFsec depicted by the dark gray half-oval.

Sec-tRNA[Ser]Sec, the elongation factor reverts to its pretRNA binding conformation, indicated by bending of the C-terminal domain downward (Figure 7B). This conformation would disfavor interaction with SBP2. Thus, whereas the SBP2-SECIS complex may remain intact once formed, this complex would recruit a new SectRNA[Ser]Sec-EFsec complex for each round of selenocysteine incorporation, supporting the second model proposed in the Introduction. A further possible scenario would invoke an SBP2 binding-dependent conformational change in the SectRNA[Ser]Sec-EFsec complex, allowing interaction with the ribosome. This further constraint would prevent SectRNA[Ser]Sec-EFsec from gaining access to an inappropriate UGA termination codon by diffusion. However, if the criteria of Sec-tRNA[Ser]Sec binding by EFsec, interaction with SBP2, and conformational changes allowing EFsec-ribosome interaction were fulfilled, codon-anticodon pairing and proofreading at the ribosomal A site would ensue. This would be followed by EFsec disengaging from the tRNA[Ser]Sec and by dissociation of EFsec

from the SBP2-SECIS complex. Empty EFsec would then reload with a new or recharged Sec-tRNA[Ser]Sec, followed by recruitment via the SBP2-SECIS complex for the next round of incorporation, as the next ribosome approaches (Figure 7B). The results reported herein provide evidence for a role of Sec-tRNA[Ser]Sec in regulating the interaction between EFsec and SBP2. The N-terminal elongation factor domain of EFsec appears to function as a negative effector of interaction with SBP2, unless tRNA[Ser]Sec is bound. Such a regulatory effect would prevent unproductive complexes between empty EFsec and SBP2, and would result in rapid dissociation of EFsec from SBP2 upon delivery of Sec-tRNA[Ser]Sec to the A site. The lack of interaction of SBP2 with full-length EFsec in the absence of tRNA[Ser]Sec, taken by itself, could be interpreted to indicate that tRNA[Ser]Sec binding in the N-terminal domain produces a conformational change that is transmitted to the C-terminal region. However, the strong stabilizing effect exerted by tRNA[Ser]Sec on the EFsec C-terminal domain when the N-terminal domain is deleted provides compelling evidence for direct contact between the C-terminal region of the elongation factor and tRNA[Ser]Sec. Thus, the C-terminal extension of EFsec may serve not only as the bridge to SBP2, but as a sensor for binding of Sec-tRNA[Ser]Sec, conveying the signal to SBP2 that interaction between the two proteins should take place. Whether, as suggested above, complex formation also affects the conformation of the SectRNA[Ser]Sec-EFsec complex and, hence, the ability to interact with the ribosome will be an intriguing subject for future investigation. Experimental Procedures Constructs Identification and characterization of the murine EFsec cDNA was described previously (Tujebajeva et al., 2000). This cDNA was cloned into the pUHD10-3 vector (Gossen and Bujard, 1992) for mammalian cell transfections. Murine SBP2 cDNA (a generous gift from Paul Copeland and Donna Driscoll) was amplified by PCR using the Expand High Fidelity PCR system (Roche, Indianapolis, IN) with primers adding NdeI and XmaI restriction sites and was then was cloned into pGBKT7 (Clontech, Palo Alto, CA). This was used as a template for in vitro translation to generate c-myc-tagged SBP2 for coimmunoprecipitation experiments. EFsec 1–305, 448–583, 469–583, and 448–520 deletion mutants were generated by PCR and then cloned into the EcoRI and BamHI sites of pGADT7 (Clontech) and used as template for in vitro translation. A glutathione-S-transferase (GST) fusion of SBP2 in an E. coli overexpression vector was constructed as follows: a PCR product containing full-length murine SBP2 was created by amplifying pcDNA3.1 SBP2 with the Expand High Fidelity PCR System using primers which added attB1 and attB2 sites 5⬘ and 3⬘, respectively. The resulting product was used to create pDONR201SBP2 using the BP reaction with the Gateway PCR system (GIBCO/Invitrogen, San Diego, CA) following the manufacturer’s instructions. This vector was then used to generate an E. coli expression vector containing an amino-terminal GST fusion of SBP2, pDEST15-SBP2, via the LR reaction of the Gateway E. coli Expression System. Similar methods were used to generate pDEST15EFsec 448–583, 469–583, 448–520, 469–520, and 520–583. Briefly, the murine EFsec cDNA was amplified with sense primers complementary to the indicated 5⬘ region containing an attB1 site, and antisense primers complementary to the indicated 3⬘ region containing an attB2 site to generate attB-containing fragments. Fragments were cloned into pDONR201 via the BP reaction of the Gateway Cloning system as above and then transferred into pDEST15 via the LR reaction to create the indicated pDEST15 EFsec deletion

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mutants. Mutations in EFsec 448–583 were introduced at positions that are highly conserved among the deduced mammalian, D. melanogaster, A. gambiae, and C. elegans EFsec amino acid sequences. Mutagenesis was carried out by PCR with overlapping oligonucleotides containing the mutation of interest and the EFsec 448–583 attB primers, and either Pfu polymerase (Stratagene, La Jolla, CA) or the Expand High Fidelity PCR System (Roche). The resulting mutant fragments were transferred into pDONR201 and then into pDEST15 as above. The GST-GUS construct was generated as a positive control for the LR reaction with the Gateway kit for pDEST15 and contains the Arabidopsis thaliana Beta glucuronidase gene. The sequences of specific oligonucleotide primers used to generate mutants or create constructs will be provided upon request. All constructs were sequenced to verify that the desired changes were introduced, with no additional changes. The human tRNA[Ser]Sec expression plasmid was a generous gift from Dolph Hatfield. In Vitro Translations and Coimmunoprecipitations In vitro transcription/translation reactions were carried out using the TnT T7 Quick Coupled Transcription/Translation System (Promega, Madison, WI) in the presence of 2 ␮l of 35S methionine (EasyTag 35S protein labeling mix, Perkin Elmer, Boston, MA) per 50 ␮l reaction for a single protein for 90 min at 30⬚C. For coimmunoprecipitations of the in vitro translated products of either full-length or truncated EFsec in pGADT7 and c-myc-tagged SBP-2 (pGBKT7 SBP2), each protein was first incubated 45 min at 30⬚C separately, then 20 ␮l of each of lysate was combined and incubated at 30⬚C for an additional 45 min, as inclusion of SBP2 and EFsec in the same reaction was found to decrease the efficiency of EFsec translation. After coincubation, 10 ␮l of each translation mix, 20 ␮l of c-myc monoclonal antibody agarose beads (Clontech), and 470 ␮l of PBS EDTA-Triton buffer (58 mM Na2HPO4, 17 mM NaH2PO4, 68 mM NaCl, 5 mM EDTA, 0.5% Triton X-100) were combined per reaction and incubated for 4 hr at 4⬚C with gentle agitation. The c-myc-agarose beads were then washed five times with 1 ml PBS, and pellets were resolved on a 10%–20% gradient SDS-PAGE gel (BioRad, Hercules, CA), which was then fixed, dried, and subjected to autoradiography. Bacterial Expression and GST Pull-Downs Bacterial expression of GST-fusion SBP2, GST-EFsec deletion mutants, and EFsec 448–583 point mutants or deletion mutants was carried out in E. coli BL21DE3 pLys S with IPTG-induction (0.2 mM final concentration, 3 hr at 37ⴗC). Cells were sonicated in extraction buffer (25 mM Tris-HCl [pH 7.5], 20 mM NaCl, 1 mM EDTA, 20% glycerol) plus protease inhibitors (Type II, Calbiochem, San Diego, CA), followed by centrifugation for 15 min at 10,000 rpm in a Sorvall RC5 at 4ⴗC to clarify. The approximate amount of GST-protein in each sample was determined by incubation of 200 ␮l crude lysate with 30 ␮l of glutathione Sepharose 4B (Amersham Pharmacia, Piscataway, NJ) for 1 hr at room temperature followed by washing three times with PBS. The pellets containing the GST-fusion protein were resolved by SDS-PAGE and Coomassie staining to determine the amount of protein expressed. For each pull-down reaction, 20 ␮l of glutathione Sepharose 4B resin was prewashed three times with 0.5 ml PBS, and approximately 30 ␮l crude E. coli GSTexpressed protein was added depending on the amount of GSTprotein expressed, with the final volume being adjusted to 30 ␮l with extraction buffer. Incubation with gentle shaking was carried out at room temperature for 1 hr, followed by addition of 10 ␮l of in vitro translated 35S methionine-labeled product and an additional 1 hr shaking at room temperature. Pellets were collected, washed three times with 0.5 ml cold IP wash buffer (25 mM Tris-HCl [pH 7.5], 300 mM NaCl, 1 mM CaCl2, 0.5% Triton X-100), and analyzed by SDS-PAGE and autoradiography. Five percent of this sample was reserved to use for Western analysis to reconfirm the amount of GST-fusion protein present. Western blotting was carried out using anti-GST antibody (Amersham Pharmacia) at 1/2000 dilution followed by horseradish peroxidase-conjugated anti-goat second antibody (Sigma, St. Louis, MO) at 1/4000 dilution and was visualized using the BM Chemilluminescence Western Blotting kit (Roche). Mammalian Cell Transfections, Coimmunoprecipitations, and Western Analysis Transient transfections in human embryonic kidney (HEK-293) cells were carried out using the calcium phosphate method of transfec-

tion as described previously (Low et al., 2000). Three days prior to transfection, cells were plated onto 60 mm culture dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were transfected with indicated amounts of pUHD10-3-based expression plasmids and 4 ␮g of the pUHD15 plasmid, which encodes a protein necessary for transcriptional activation of the pUHD10-3 promoter (Gossen and Bujard, 1992). To monitor transfection efficiencies, cells were cotransfected with 3 ␮g of an expression vector containing the human growth hormone cDNA under control of the HSV thymidine kinase promoter. Coimmunoprecipitation of SBP2 from cell lysates and visualization of FLAG-tagged EFsec or EFsec 386–583 by Western analysis was performed as previously described (Tujebajeva et al., 2000). Type 1 deiodinase assays were carried out as described previously (Low et al., 2000). Pulse-Labeling of Proteins with 35S Methionine and Cysteine HEK-293 cells were transfected as described above. Two days after transfection, media were changed to cysteine and methionine-free DMEM (Sigma) without serum for 1 hr, followed by incubation for 1 hr with EasyTag 35S protein labeling mix (35S cysteine and methionine, Perkin Elmer) at 100 ␮Ci/ml. After labeling, cells were incubated in DMEM plus 10% FBS for the indicated times and then harvested in IP wash buffer plus type III protease inhibitors (Calbiochem). Samples were lysed by freeze/thawing three times, and debris was pelleted by microcentrifugation. Supernatants were precleared with protein A/G agarose (Oncogene/Calbiochem, San Diego, CA) for 20 min on ice and incubated with 3 ␮l of anti-FLAG antibody overnight at 4⬚C, followed by incubation with 20 ␮l of protein A/G agarose for 2 hr at 4⬚C. Pellets were collected by centrifugation and washed once in IP wash buffer and once in IP wash buffer containing 14 mM NaCl. Pellets were resolved on a 10%–20% SDS-PAGE gel, which was then fixed, dried, and autoradiographed. Acknowledgments This work was supported by the NIH grant DK47320. The authors wish to thank Matthias Hentze, Dolph Hatfield, and Dennis Dowhan for critical reading and helpful suggestions on the manuscript. Received: November 7, 2002 Revised: January 9, 2003 References Atkins, J.F., Bock, A., Matsufuji, S., and Gesteland, R.F. (1999). Dynamics of the genetic code. In The RNA World, R.F. Gesteland, T.R. Cech, and J.F. Atkins, eds. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 637–673. Baron, C., Heider, J., and Bock, A. (1993). Interaction of translation factor SELB with the formate dehydrogenase H selenopolypeptide mRNA. Proc. Natl. Acad. Sci. USA 90, 4181–4185. Berry, M.J., Banu, L., Harney, J.W., and Larsen, P.R. (1993). Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons. EMBO J. 12, 3315– 3322. Berry, M.J., Harney, J.W., Ohama, T., and Hatfield, D.L. (1994). Selenocysteine insertion or termination: factors affecting UGA codon fate and complementary anticodon:codon mutations. Nucleic Acids Res. 22, 3753–3759. Copeland, P.R., Fletcher, J.E., Carlson, B.A., Hatfield, D.L., and Driscoll, D.M. (2000). A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs. EMBO J. 19, 306–314. Fagegaltier, D., Hubert, N., Yamada, K., Mizutani, T., Carbon, P., and Krol, A. (2000). Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation. EMBO J. 19, 4796– 4805. Gossen, M., and Bujard, H. (1992). Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551. Low, S.C., Grundner-Culemann, E., Harney, J.W., and Berry, M.J.

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(2000). SECIS-SBP2 interactions dictate selenocysteine incorporation efficiency and selenoprotein hierarchy. EMBO J. 19, 6882–6890. Moriarty, P.M., Reddy, C.C., and Maquat, L.E. (1998). Selenium deficiency reduces the abundance of mRNA for Se-dependent glutathione peroxidase 1 by a UGA-dependent mechanism likely to be nonsense codon-mediated decay of cytoplasmic mRNA. Mol. Cell. Biol. 18, 2932–2939. Thanbichler, M., Bock, A., and Goody, R.S. (2000). Kinetics of the interaction of translation factor SelB from Escherichia coli with guanosine nucleotides and selenocysteine insertion sequence RNA. J. Biol. Chem. 275, 20458–20466. Tujebajeva, R.M., Copeland, P.R., Xu, X.M., Carlson, B.A., Harney, J.W., Driscoll, D.M., Hatfield, D.L., and Berry, M.J. (2000). Decoding apparatus for eukaryotic selenocysteine incorporation. EMBO Rep. 2, 158–163. Weiss, S.L., and Sunde, R.A. (1998). Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels. RNA 4, 816–827.