Functional Analysis of the Interplay between Translation Termination, Selenocysteine Codon Context, and Selenocysteine Insertion Sequence-binding Protein 2
2007; Elsevier BV; Volume: 282; Issue: 51 Linguagem: Inglês
10.1074/jbc.m707061200
ISSN1083-351X
AutoresMalavika Gupta, Paul R. Copeland,
Tópico(s)Trace Elements in Health
ResumoA selenocysteine insertion sequence (SECIS) element in the 3′-untranslated region and an in-frame UGA codon are the requisite cis-acting elements for the incorporation of selenocysteine into selenoproteins. Equally important are the trans-acting factors SBP2, Sec-tRNA[Ser]Sec, and eEFSec. Multiple in-frame UGAs and two SECIS elements make the mRNA encoding selenoprotein P (Sel P) unique. To study the role of codon context in determining the efficiency of UGA readthrough at each of the 10 rat Sel P Sec codons, we individually cloned 27-nucleotide-long fragments representing each UGA codon context into a luciferase reporter construct harboring both Sel P SECIS elements. Significant differences, spanning an 8-fold range of UGA readthrough efficiency, were observed, but these differences were dramatically reduced in the presence of excess SBP2. Mutational analysis of the "fourth base" of contexts 1 and 5 revealed that only the latter followed the established rules for hierarchy of translation termination. In addition, mutations in either or both of the Sel P SECIS elements resulted in differential effects on UGA readthrough. Interestingly, even when both SECIS elements harbored a mutation of the core region required for Sec incorporation, context 5 retained a significantly higher level of readthrough than context 1. We also show that SBP2-dependent Sec incorporation is able to repress G418-induced UGA readthrough as well as eRF1-induced stimulation of termination. We conclude that a large codon context forms a cis-element that works together with Sec incorporation factors to determine readthrough efficiency. A selenocysteine insertion sequence (SECIS) element in the 3′-untranslated region and an in-frame UGA codon are the requisite cis-acting elements for the incorporation of selenocysteine into selenoproteins. Equally important are the trans-acting factors SBP2, Sec-tRNA[Ser]Sec, and eEFSec. Multiple in-frame UGAs and two SECIS elements make the mRNA encoding selenoprotein P (Sel P) unique. To study the role of codon context in determining the efficiency of UGA readthrough at each of the 10 rat Sel P Sec codons, we individually cloned 27-nucleotide-long fragments representing each UGA codon context into a luciferase reporter construct harboring both Sel P SECIS elements. Significant differences, spanning an 8-fold range of UGA readthrough efficiency, were observed, but these differences were dramatically reduced in the presence of excess SBP2. Mutational analysis of the "fourth base" of contexts 1 and 5 revealed that only the latter followed the established rules for hierarchy of translation termination. In addition, mutations in either or both of the Sel P SECIS elements resulted in differential effects on UGA readthrough. Interestingly, even when both SECIS elements harbored a mutation of the core region required for Sec incorporation, context 5 retained a significantly higher level of readthrough than context 1. We also show that SBP2-dependent Sec incorporation is able to repress G418-induced UGA readthrough as well as eRF1-induced stimulation of termination. We conclude that a large codon context forms a cis-element that works together with Sec incorporation factors to determine readthrough efficiency. Selenocysteine (Sec), 2The abbreviations used are:SecselenocysteineSBP2Sec insertion sequence binding protein 2UTRuntranslated regionRRLrabbit reticulocyte lysate. the 21st amino acid, is incorporated into proteins via a recoding of the UGA stop codon (1Copeland P.R. Gene (Amst.). 2003; 312: 17-25Crossref PubMed Scopus (63) Google Scholar). Selenoproteins are primarily involved in protecting the cell from oxidative stress, and the concerted effort of several protein factors and RNA elements is required for the production of these proteins (2Driscoll D.M. Copeland P.R. Annu. Rev. Nutr. 2003; 23: 17-40Crossref PubMed Scopus (321) Google Scholar). Two cis-elements in the mRNA are required for the incorporation of selenocysteine into a nascent polypeptide. These are an in-frame UGA codon and a structure called the SECIS (Sec insertion sequence) element (3Caban K. Copeland P.R. Cell Mol. Life Sci. 2006; 63: 73-81Crossref PubMed Scopus (43) Google Scholar). The SECIS element is a stem loop structure consisting of a core stem and a terminal bulge or loop. The SECIS core is comprised of an AUGA motif positioned opposite a GA dinucleotide forming a non-Watson-Crick base-paired quartet, thus making this RNA a member of the kink-turn family of RNA motifs (3Caban K. Copeland P.R. Cell Mol. Life Sci. 2006; 63: 73-81Crossref PubMed Scopus (43) Google Scholar). The terminus of the SECIS stem consists of either a 9–11-nucleotide loop (designated Form 1) or a 5′ bulge followed by a smaller 6-nucleotide loop (designated Form 2). Both SECIS forms contain a conserved AAR motif within the loop or bulge, respectively (4Grundner-Culemann E. Martin 3rd, G.W. Harney J.W. Berry M.J. RNA (N. Y.). 1999; 5: 625-635Crossref PubMed Scopus (129) Google Scholar). SECIS binding protein 2 (SBP2) (5Copeland P.R. Fletcher J.E. Carlson B.A. Hatfield D.L. Driscoll D.M. EMBO J. 2000; 19: 306-314Crossref PubMed Scopus (314) Google Scholar), a Sec-specific translation elongation factor eEFSec (6Berry M.J. Tujebajeva R.M. Copeland P.R. Xu X.M. Carlson B.A. Martin 3rd, G.W. Low S.C. Mansell J.B. Grundner-Culemann E. Harney J.W. Driscoll D.M. Hatfield D.L. Biofactors. 2001; 14: 17-24Crossref PubMed Scopus (57) Google Scholar), and a Sec-tRNA[Ser]Sec (7Lee B.J. Rajagopalan M. Kim Y.S. You K.H. Jacobson K.B. Hatfield D. Mol. Cell. Biol. 1990; 10: 1940-1949Crossref PubMed Scopus (106) Google Scholar) are the three trans-acting factors that have been identified to be essential for Sec incorporation. Recently ribosomal protein L30 has been found to interact with the SECIS element but whether it is required for Sec incorporation remains to be determined (8Chavatte L. Brown B.A. Driscoll D.M. Nat. Struct. Mol. Biol. 2005; 12: 408-416Crossref PubMed Scopus (147) Google Scholar). selenocysteine Sec insertion sequence binding protein 2 untranslated region rabbit reticulocyte lysate. Most selenoprotein mRNAs contain only a single Sec codon and a single SECIS element. The selenoprotein P (Sel P) mRNA is unique because it contains multiple Sec codons ranging from 10 in mammals to a maximum of 18 in some amphibians (9Berry M.J. Nat. Struct. Mol. Biol. 2005; 12: 389-390Crossref PubMed Scopus (16) Google Scholar, 10Hill K.E. Lloyd R.S. Yang J.G. Read R. Burk R.F. J. Biol. Chem. 1991; 266: 10050-10053Abstract Full Text PDF PubMed Google Scholar), and it has two SECIS elements (11Hill K.E. Lloyd R.S. Burk R.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 537-541Crossref PubMed Scopus (161) Google Scholar). In addition to termination at its naturally occurring stop codon, rat Sel P has protein isoforms resulting from termination at the second, third, and seventh UGAs (12Ma S. Hill K.E. Caprioli R.M. Burk R.F. J. Biol. Chem. 2002; 277: 12749-12754Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The in vivo frequency of translation termination at these UGAs is not known, but these isoforms have been observed in preparations of Sel P purified from rat serum (12Ma S. Hill K.E. Caprioli R.M. Burk R.F. J. Biol. Chem. 2002; 277: 12749-12754Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The role of the dual SECIS elements has only recently been studied in detail. In 2006 Stoytcheva et al. (13Stoytcheva Z. Tujebajeva R.M. Harney J.W. Berry M.J. Mol. Cell. Biol. 2006; 26: 9177-9184Crossref PubMed Scopus (51) Google Scholar) determined that the second SECIS element of zebrafish Sel P was required for readthrough of the first UGA and the first SECIS element was necessary for readthrough of subsequent UGAs and consequently, production of full-length protein (13Stoytcheva Z. Tujebajeva R.M. Harney J.W. Berry M.J. Mol. Cell. Biol. 2006; 26: 9177-9184Crossref PubMed Scopus (51) Google Scholar). However, the mechanistic basis for this distinction of function remains elusive. Studies in bacteria and eukaryotes have shown that translation termination is influenced by the bases near the stop codon, with a strong bias toward nucleotides 3′ of the stop codon (14Mottagui-Tabar S. Bjornsson A. Isaksson L.A. EMBO J. 1994; 13: 249-257Crossref PubMed Scopus (111) Google Scholar, 15Namy O. Hatin I. Rousset J.P. EMBO Rep. 2001; 2: 787-793Crossref PubMed Scopus (159) Google Scholar, 16Poole E.S. Brown C.M. Tate W.P. EMBO J. 1995; 14: 151-158Crossref PubMed Scopus (214) Google Scholar). These findings support the model that translation termination is dictated by more than three residues with the identity of the base at the fourth position (+4 position; hereafter referred to as the "fourth base") contributing to the efficiency of translation termination. In several organisms including Escherichia coli and mammals, the strength of a translation termination context follows the order of A > G > C > U, where an mRNA with a stop codon followed by an A is terminated more efficiently than one with a U at the same position (16Poole E.S. Brown C.M. Tate W.P. EMBO J. 1995; 14: 151-158Crossref PubMed Scopus (214) Google Scholar, 17Cridge A.G. Major L.L. Mahagaonkar A.A. Poole E.S. Isaksson L.A. Tate W.P. Nucleic Acids Res. 2006; 34: 1959-1973Crossref PubMed Scopus (44) Google Scholar, 18McCaughan K.K. Brown C.M. Dalphin M.E. Berry M.J. Tate W.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5431-5435Crossref PubMed Scopus (228) Google Scholar). More recent studies have shown that translation termination is determined by a combination of the identity of the stop codon and the nucleotide at the fourth base (16Poole E.S. Brown C.M. Tate W.P. EMBO J. 1995; 14: 151-158Crossref PubMed Scopus (214) Google Scholar, 17Cridge A.G. Major L.L. Mahagaonkar A.A. Poole E.S. Isaksson L.A. Tate W.P. Nucleic Acids Res. 2006; 34: 1959-1973Crossref PubMed Scopus (44) Google Scholar, 19Manuvakhova M. Keeling K. Bedwell D.M. RNA (N. Y.). 2000; 6: 1044-1055Crossref PubMed Scopus (297) Google Scholar). Studies have shown the ratio of Sec incorporation to termination also to be affected by the base immediately 3′ of the Sec codon. The rules for termination at a Sec codon differ from the A > G > C > U rule observed in non-Sec codons in mammals and E. coli. A study examining the effect of the fourth base on the porcine phospholipid hydroperoxide glutathione peroxidase UGA readthrough found it to be highest with the native fourth base, a G (20Nasim M.T. Jaenecke S. Belduz A. Kollmus H. Flohe L. McCarthy J.E.G. J. Biol. Chem. 2000; 275: 14846-14852Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). A gradual decrease in readthrough was observed when the fourth base was mutated to a C or a U. An A at the fourth base of the same construct resulted in the most drastic reduction in readthrough (20Nasim M.T. Jaenecke S. Belduz A. Kollmus H. Flohe L. McCarthy J.E.G. J. Biol. Chem. 2000; 275: 14846-14852Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). A separate study of Sec incorporation in a Type 1 deiodinase reporter showed that when the Sec codon was followed by a strong termination context, an A as the fourth base, readthrough was much greater than when the codon was followed by any of the remaining three bases (21Grundner-Culemann E. Martin G.W. Tujebajeva R. Harney J.W. Berry M.J. J. Mol. Biol. 2001; 310: 699-707Crossref PubMed Scopus (23) Google Scholar). These previous studies have contributed to the model that the efficiency of Sec incorporation is not simply inversely proportional to the efficiency of translation termination, suggesting that other determinants are required for processive and efficient Sec incorporation. Previously we showed that in vitro, Sec incorporation in a monocistronic luciferase reporter bearing the rat phospholipid hydroperoxide glutathione peroxidase SECIS element occurs with a maximum of ∼8% efficiency. This contrasted with an ∼40% efficiency when full-length Sel P was translated in the same system, suggesting that one or more elements within the Sel P mRNA sequence were providing increased efficiency (22Mehta A. Rebsch C.M. Kinzy S.A. Fletcher J.E. Copeland P.R. J. Biol. Chem. 2004; 279: 37852-37859Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). This study determines how Sel P sequences contribute to the efficiency of Sec incorporation and its interplay with translation termination. Codon Context Plasmid Construction—A firefly luciferase construct containing an in-frame UGA at position 258 was created as previously described (22Mehta A. Rebsch C.M. Kinzy S.A. Fletcher J.E. Copeland P.R. J. Biol. Chem. 2004; 279: 37852-37859Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The coding region of this construct was further mutated, 12 nucleotides upstream and downstream of UGA 258, to create AflIII and SgfI restriction sites. PacI and NotI linkers were used to clone the first 770 nucleotides of the Sel P 3′-UTR downstream of the Luc UGA open reading frame. This stretch of 3′-UTR contained both SECIS elements. Oligos, 27 nucleotides long and containing each Sel P UGA codon and its context (12 nucleotides flanking on either side), were synthesized with AflIII and SgfI linkers. These oligos were then phosphorylated and ligated into the firefly luciferase vector pre-digested with AflIII and SgfI. The reading frame of the original firefly luciferase construct was not shifted by cloning the 27-nucleotide Sel P oligos. All constructs were in pcDNA3.1 (Invitrogen) and regulated by the bacteriophage T7 promoter. Generation of Point Mutants—Point mutants were created using site-directed mutagenesis (QuikChange, Stratagene) as per the manufacturer's protocol. Mutants were verified by automated DNA sequencing. In Vitro Transcription and Translation—Constructs were linearized with either XhoI or PacI. XhoI digestion generated a template with a 3′-UTR downstream of the T7 promoter, allowing transcription of the coding region and the 770 nucleotides of the Sel P 3′-UTR. PacI digestion created a template that had the 3′-UTR, including the SECIS elements, upstream of the T7 promoter, thereby allowing transcription of only the coding region and not the 3′-UTR. The eRF1 and eEFSec constructs were digested with XbaI and HindIII, respectively. Linearized DNA was then transcribed with the T7 mMessage kit (Ambion). Rabbit reticulocyte lysate (Promega) was used for in vitro translations. In vitro translations were performed with [35S]Met according to the manufacturer's protocol and three separate translations were done for each construct. Briefly, 12.5-μl reactions contained 8 μl of rabbit reticulocyte lysate (RRL), 0.25 μl of 40 units/μl of RNasin (Promega), 0.25 μl of 1 mm amino acid mixture minus methionine, 0.5 μl of [35S]Met at 10 mCi/ml, 100 ng of reporter mRNA and varying amounts of CTSBP2 or 1× phosphate-buffered saline and 2 mm dithiothreitol. The amount of CTSBP2 added to the reactions was either 0.2 or 2 pmol, but the total volume of CTSBP2 was 3 μl. CTSBP2 was diluted in 1× phosphate-buffered saline and 2 mm dithiothreitol. Two microliters of the translation reactions were resolved by 12% SDS-PAGE and quantitated by PhosphorImager analysis. As translations carried out in the absence of added SBP2 yielded a full-length product of low intensity, these reactions were exposed for up to 8 days. An overnight exposure was sufficient for reactions carried out with CTSBP2. To quantitate the bands, a rectangle or a polygon was drawn around each full-length and UGA-termination product. The background was subtracted by copying and pasting the rectangles or polygons directly above our product of interest. For co-translation experiments, 25 ng of human eRF1 and 100 ng of mouse eEFSec mRNAs were translated with the specified Sel P contexts. 75Se Labeling—Selenium 75 (400 μm at 380 mCi/mg), in the form of selenous acid (obtained from the University of Missouri Research Reactor) was first neutralized with NaOH and diluted to a working concentration of 0.8 μm. The final concentration of 75Se in our reactions was 0.064 μm. Except for the addition of 75Se, in vitro translations were carried out exactly as explained above. After the 1-h incubation, 16% of the translation reactions were resolved by 12% SDS-PAGE and quantitated by PhosphorImager analysis. To account for potential differences in sample loading, the SDS gel was stained with Gel Code Blue (Pierce) and an unknown protein consistently present in each sample was used to normalize the 75Se-labeled product. Preparation of CTSBP2—Recombinant Xpress/His-tagged CTSBP2 was made as previously described (23Kinzy S.A. Caban K. Copeland P.R. Nucleic Acids Res. 2005; 33: 5172-5180Crossref PubMed Scopus (43) Google Scholar). Bioinformatic Analysis—Mouse, human, bovine, and orangutan Sel P Sec codons and their 12 flanking nucleotides on either side were aligned to the corresponding conserved Sec codons and their contexts in rat Sel P. WebLogo was used to graph homology (29Crooks G.E. Hon G. Chandonia J.M. Brenner S.E. Genome Res. 2004; 14: 1188-1190Crossref PubMed Scopus (8442) Google Scholar). Sel P Codon Contexts—To understand the impact of codon context on Sec incorporation, we used each of the 10 rat Sel P UGAs, along with their four flanking codons on both sides, replacing the corresponding sequence in the open reading frame of a firefly luciferase construct (Fig. 1, Sec1–Sec10). This monocistronic luciferase construct contained an in-frame UGA at codon 258 (Fig. 1, Luc UGA). A Sec codon at this position had been previously shown to permit Sec incorporation at an efficiency of ∼5–8% (22Mehta A. Rebsch C.M. Kinzy S.A. Fletcher J.E. Copeland P.R. J. Biol. Chem. 2004; 279: 37852-37859Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In addition to the Sel P UGA codons and their native codon contexts, our constructs also contained the first 770 nucleotides of the rat Sel P 3′-UTR including both of its SECIS elements (Fig. 1). In rat Sel P, the seventh to the 10th Sec codons are closely clustered. Consequently, Sec constructs 7–10 contained more than one inframe UGA and common or overlapping nucleotides. The overlapping UGAs, which are underlined in Fig. 1, were mutated to the UGU cysteine codon, thus generating constructs designated Sec UGU. Sec10 was further mutated to change the natural rat Sel P stop codon, UAA, to the tyrosine UAU codon, enabling us to assess its readthrough ability. As a control, we used the parent firefly luciferase construct with the in-frame UGA at amino acid 258 (Fig. 1, Luc UGA). Like the Sec constructs, the Luc UGA construct also contained the first 770 nucleotides of the rat Sel P 3′-UTR including both of the rat Sel P SECIS elements. Because RRL contains all of the factors so far identified to be required for Sec incorporation (5Copeland P.R. Fletcher J.E. Carlson B.A. Hatfield D.L. Driscoll D.M. EMBO J. 2000; 19: 306-314Crossref PubMed Scopus (314) Google Scholar, 22Mehta A. Rebsch C.M. Kinzy S.A. Fletcher J.E. Copeland P.R. J. Biol. Chem. 2004; 279: 37852-37859Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), we used this system to assess any differences in readthrough of the Sel P codon contexts. As expected, all of our in vitro translation reactions produced two predominant products. The larger product corresponded to the full-length polypeptide resulting from UGA readthrough, whereas the smaller product was the pre-Sec peptide resulting from termination at the UGA codon. The efficiency of translation for each construct was reported as the percent readthrough calculated by dividing the amount of fulllength polypeptide produced by the sum of the full-length and pre-Sec peptides. Most of the experiments make use of [35S]Met to label both termination and readthrough products so that we can evaluate both nonspecific readthrough as well as that resulting from Sec incorporation. Because Sec incorporation in RRL is limiting for SBP2 (22Mehta A. Rebsch C.M. Kinzy S.A. Fletcher J.E. Copeland P.R. J. Biol. Chem. 2004; 279: 37852-37859Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), all of our in vitro translations were carried out either in the absence of added SBP2 or with recombinant rat SBP2 corresponding to the fully functional C-terminal 445 amino acids and bearing an N-terminal Xpress/His tag (CTSBP2) (22Mehta A. Rebsch C.M. Kinzy S.A. Fletcher J.E. Copeland P.R. J. Biol. Chem. 2004; 279: 37852-37859Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The amount of CTSBP2 needed to reach intermediate (0.2 pmol) and saturating (2 pmol) levels of Sec incorporation was experimentally determined by using varying amounts of SBP2 in the luciferase-based Sec incorporation assay (data not shown). Assessing the efficiency of Sec incorporation in the absence of added CTSBP2 presented a challenge in that long exposures were required to acquire accurate quantitation of the readthrough product. These exposures resulted in a saturation of signal for the termination product and thus in these cases the value for efficiency was slightly overestimated. When the full-length products were normalized by comparing them to that obtained for the Luc UGA control, we calculated the level of overestimation to be a factor of 1.4 (data not shown). Sec Incorporation in Sel P Is Regulated by Codon Context—Each Sel P codon context was first in vitro translated in the absence of added SBP2, thus making use of the limiting amount of endogenous SBP2 in RRL (5Copeland P.R. Fletcher J.E. Carlson B.A. Hatfield D.L. Driscoll D.M. EMBO J. 2000; 19: 306-314Crossref PubMed Scopus (314) Google Scholar). Of all the Sel P contexts with a single Sec codon, Sec1 might be expected to have the greatest readthrough because it has what is classically described as the weakest context for translation termination, i.e. a U as the fourth base (Fig. 1). Yet, based on our in vitro translation data, Sec1 provided lower readthrough than Sec4–Sec6, each of which has a stronger predicted context for termination with a C at the fourth base (Fig. 2A, compare lane 2 with lanes 5–7). In the absence of CTSBP2, several contexts provided virtually no readthrough including the Luc UGA control, Sec3, and Sec7–Sec10. Interestingly, significant differences in readthrough efficiency were observed among Sec3–Sec6, all of which share a C residue at the fourth base. Sec3 was ∼5-fold less efficient at readthrough than Sec6 and 8-fold less efficient than Sec4 and Sec5 (Fig. 2A compare lanes 4–7). The striking lack of readthrough for Sec7–Sec10 with endogenous SBP2 is likely due to the presence of dual UGA codons in each of these contexts. A graphical representation of the amount of readthrough supported by the Sel P UGA codon contexts is shown in Fig. 2C (white bars). Fig. 2, A and C, clearly show that the Sel P Sec codon contexts had readthrough efficiencies that did not correlate with the fourth base hierarchy for translation termination. These results, together with previous studies of the role of the fourth base in regulating Sec incorporation, confirm that the fourth base of a Sec codon context is not a good predictor of readthrough efficiency thus alluding to the involvement of either a larger codon context determinant, a role for the SECIS elements, or both. To analyze the differences in readthrough efficiency under conditions where Sec incorporation levels were increased, we supplemented the translation reactions with 0.2 pmol of CTSBP2, which is in the middle of the linear range of response for the in vitro Sec incorporation assay (data not shown). Under these conditions, there was a 3–16-fold increase in readthrough for most constructs (Fig. 2B) and a general reduction in the differences in readthrough efficiency between constructs (Fig. 2C, gray bars). The Luc UGA control appears to generate a higher level of full-length product, but the amount of termination product is consistently higher. The basis for this, as well as the fact that the pre-Sec peptide runs at a slightly lower apparent molecular weight, is unknown. With 0.2 pmol of CTSBP2, average readthrough of Sec1 rose from 3.1 to 10% (compare lane 2 in Fig. 2, A and B), whereas average readthrough for Sec3 rose almost 16-fold from 0.7 to 11% (compare lane 4 in Fig. 2, A and B). Sec4–Sec6, all of which had the highest average readthrough with endogenous SBP2, maintained their high readthrough profile in the presence of 0.2 pmol of CTSBP2. Average readthrough of Sec4 and Sec5 rose 3-fold from ∼5 to ∼15%, respectively, whereas average readthrough of Sec6 rose 4-fold from 3 to 13% (compare Fig. 2, A, lanes 5–7 to B, lanes 5–7). These data are shown graphically in Fig. 2C (gray bars). Interestingly, in the contexts that contained a single Sec codon, the differences in readthrough that existed in the presence of endogenous SBP2 or 0.2 pmol of CTSBP2 were eliminated with saturating levels of SBP2. When saturating levels of SBP2 (2 pmol of CTSBP2) were added to the reactions, readthrough was again substantially increased in the constructs containing a single in-frame Sec codon (Sec1–Sec6) but to a lesser extent in the dual UGA contexts (Sec7–Sec10). Fig. 2C (black bars) shows that with saturating amounts of SBP2, average readthrough reaches its peak with an efficiency of ∼20%, still below the maximum of 40% achieved with full-length Sel P in vitro (22Mehta A. Rebsch C.M. Kinzy S.A. Fletcher J.E. Copeland P.R. J. Biol. Chem. 2004; 279: 37852-37859Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The contexts containing two Sec codons (Sec7–Sec10) yielded uniformly lower readthrough both in the presence and absence of CTSBP2 when compared with all other contexts (Fig. 2, A and B, lanes 8–11). With saturating amounts of SBP2, readthrough of Sec7–Sec10 was still restricted to ∼7% (Fig. 2C, black bars). That excess SBP2 could not promote maximum Sec incorporation efficiency (∼20%; Fig. 2C, black bars) suggested that the decoding of dual UGA codons as Sec may require other cis-elements present in full-length Sel P. Sel P SECIS Elements Differentially Regulate Readthrough and Sec Incorporation—Because the addition of SBP2 to RRL reduced the differences in readthrough dictated by codon context, we set out to determine the specific contributions of readthrough resulting from Sec incorporation versus nonspecific readthrough (translational infidelity) using Sec1 and Sec5 as examples of low and high efficiency, respectively. As a control to test for UGA specificity, we mutated the Sec codon of Sec1 and Sec5 to the UAA stop codon. This nonsense mutation yielded no readthrough even in the presence of added SBP2 showing our readthrough was UGA-specific (data not shown). In addition, to assess the potential role of eEFsec in regulating Sec incorporation efficiency, Sec1 and Sec5 mRNAs were co-translated with 100 ng of eEFSec mRNA, which had no effect on readthrough (data not shown). This result was not unexpected because we have previously established that RRL is limiting only for SBP2 (22Mehta A. Rebsch C.M. Kinzy S.A. Fletcher J.E. Copeland P.R. J. Biol. Chem. 2004; 279: 37852-37859Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). To create constructs that were unable to support Sec incorporation, both SECIS elements were mutated at the core region (AUGA to AUCC), a mutation that eliminates the 5′ side of the essential GA quartet and that prevents SBP2 binding and Sec incorporation for other SECIS elements (24Lesoon A. Mehta A. Singh R. Chisolm G.M. Driscoll D.M. Mol. Cell. Biol. 1997; 17: 1977-1985Crossref PubMed Scopus (75) Google Scholar). 3N. Rodriguez and P. R. Copeland, unpublished results. Fig. 3A (lanes 4, 5, 9, and 10) shows the results of translating Sec1 and Sec5 mRNAs harboring double mutant SECIS elements (M1M2) with endogenous SBP2 or 0.2 pmol of CTSBP2. With endogenous SBP2, the double mutation in Sec5 decreased readthrough from ∼5 to ∼3% (Fig. 3A, compare lanes 3 and 5), indicating that the efficiency of Sec incorporation is only about 2% (Fig. 3C, white bars). In the case of Sec1, with endogenous SBP2, the double SECIS mutants reduced readthrough from ∼3 to ∼1% (Fig. 3A, compare lanes 2 and 4) thus indicating the same relative amount of Sec incorporation as found for Sec5 (Fig. 3C, white bars). As expected, the addition of 0.2 pmol of CTSBP2 had no stimulatory effect on readthrough for either Sec1 M1M2 or Sec5 M1M2 (Fig. 3A, compare lanes 4 and 5 to lanes 9 and 10). That Sec5 M1M2 retained significantly higher readthrough relative to Sec1 M1M2 indicates that most of the readthrough apparent for Sec5, in the absence of added SBP2, is the result of translation infidelity as opposed to Sec incorporation. Table 1 shows the amount of readthrough due to Sec incorporation for Sec1 and Sec5 calculated as the difference between readthrough observed with wild-type SECIS elements and that observed with the double SECIS mutants. These results reiterate that most of the readthrough obtained for Sec5 in the absence of added SBP2 is the result of translational infidelity. However, this activity is suppressed in the presence of saturating levels of SBP2, suggesting that when Sec incorporation is relatively efficient, it is able to suppress nonspecific readthrough of UGA codons.TABLE 1Absolute percentage of Sec incorporation of Sec1 and Sec5 (when translated with 0, 0.2, or 2 pmol of CTSBP2) as calculated by subtracting readthrough of their respective double SECIS mutants (Sec1-Sec1 M1M2 or Sec5-Sec5 M1M2)ConstructAbsolute % of Sec incorporation±S.E.Sec1; 0 pmol of CTSBP22.190.29Sec5; 0 pmol of CTSBP22.340.7Sec1; 0.2 pmol of CTSBP290.7Sec5; 0.2 pmol of CTSBP212.850.79Sec1; 2 pmol of CTSBP2190.97Sec5; 2 pmol of CTSBP2200.7 Open table in a new tab
Referência(s)