Recoding elements located adjacent to a subset of eukaryal selenocysteine-specifying UGA codons
2005; Springer Nature; Volume: 24; Issue: 8 Linguagem: Inglês
10.1038/sj.emboj.7600642
ISSN1460-2075
AutoresMichael Howard, Gaurav Aggarwal, Christine B. Anderson, Shikha Khatri, Kevin M. Flanigan, John F. Atkins,
Tópico(s)Selenium in Biological Systems
ResumoArticle24 March 2005free access Recoding elements located adjacent to a subset of eukaryal selenocysteine-specifying UGA codons Michael T Howard Corresponding Author Michael T Howard Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Gaurav Aggarwal Gaurav Aggarwal Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Christine B Anderson Christine B Anderson Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Shikha Khatri Shikha Khatri Department of Human Genetics, University of Utah, Salt Lake City, UT, USAPresent address: Genome Research Institute, University of Cincinnati, Bldg D Room 227, 2180 E Galbraith Road, Cincinnati, OH 45237, USA Search for more papers by this author Kevin M Flanigan Kevin M Flanigan Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Departments of Neurology and Pathology, University of Utah, Salt Lake City, UT, USA Search for more papers by this author John F Atkins John F Atkins Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Bioscience Institute, University College Cork, Cork, Ireland Search for more papers by this author Michael T Howard Corresponding Author Michael T Howard Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Gaurav Aggarwal Gaurav Aggarwal Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Christine B Anderson Christine B Anderson Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Shikha Khatri Shikha Khatri Department of Human Genetics, University of Utah, Salt Lake City, UT, USAPresent address: Genome Research Institute, University of Cincinnati, Bldg D Room 227, 2180 E Galbraith Road, Cincinnati, OH 45237, USA Search for more papers by this author Kevin M Flanigan Kevin M Flanigan Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Departments of Neurology and Pathology, University of Utah, Salt Lake City, UT, USA Search for more papers by this author John F Atkins John F Atkins Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Bioscience Institute, University College Cork, Cork, Ireland Search for more papers by this author Author Information Michael T Howard 1, Gaurav Aggarwal1, Christine B Anderson1, Shikha Khatri1, Kevin M Flanigan1,2 and John F Atkins1,3 1Department of Human Genetics, University of Utah, Salt Lake City, UT, USA 2Departments of Neurology and Pathology, University of Utah, Salt Lake City, UT, USA 3Bioscience Institute, University College Cork, Cork, Ireland *Corresponding author. Department of Human Genetics, University of Utah, 15 N 2030 E, Rm 2100, Salt Lake City, UT 84112, USA. Tel.: +1 801 585 1927; Fax: +1 801 585 3910; E-mail: [email protected] The EMBO Journal (2005)24:1596-1607https://doi.org/10.1038/sj.emboj.7600642 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Incorporation of the 21st amino acid, selenocysteine, into proteins is specified in all three domains of life by dynamic translational redefinition of UGA codons. In eukarya and archaea, selenocysteine insertion requires a cis-acting selenocysteine insertion sequence (SECIS) usually located in the 3′UTR of selenoprotein mRNAs. Here we present comparative sequence analysis and experimental data supporting the presence of a second stop codon redefinition element located adjacent to a selenocysteine-encoding UGA codon in the eukaryal gene, SEPN1. This element is sufficient to stimulate high-level (6%) translational redefinition of the SEPN1 UGA codon in human cells. Readthrough levels further increased to 12% when tested in the presence of the SEPN1 3′UTR SECIS. Directed mutagenesis and phylogeny of the sequence context strongly supports the importance of a stem loop starting six nucleotides 3′ of the UGA codon. Sequences capable of forming strong RNA structures were also identified 3′ adjacent to, or near, selenocysteine-encoding UGA codons in the Sps2, SelH, SelO, and SelT selenoprotein genes. Introduction Dynamic reprogramming of the genetic code redefines a subset of stop codons to encode an amino acid. Translation beyond the stop codon results in a fusion protein derived from information in two adjacent open reading frames. In some cases, continued translation beyond the stop codon is the relevant feature and the identity of the specified amino acid unimportant (Namy et al, 2004). In other cases, the identity of the specified amino acid is critical. Redefinition of UGA codons to specify the 21st amino acid, selenocysteine, directs insertion of this highly reactive amino acid, which is often a required residue for protein activity (for reviews, see Hatfield and Gladyshev, 2002; Driscoll and Copeland, 2003). The recoding of stop codons in select mRNAs discussed in this manuscript is to be clearly distinguished from the genome-wide reassignment of stop codons found in mycoplasma, ciliates, and some mitochondria, where the reassigned codon is directly decoded in all mRNAs as a sense codon. The extension of the genetic code to specify UGA decoding as selenocysteine is found in a subset of genes with diverse functions in all three domains of life (reviewed in Hatfield and Gladyshev, 2002). In eukarya and archaea, decoding UGA as selenocysteine requires a selenocysteine insertion sequence (SECIS) located in the 3′UTR of most selenoprotein-encoding mRNAs (Berry et al, 1991; Rother et al, 2001). In contrast, bacterial bSECIS elements are located immediately adjacent to the UGA codon (Zinoni et al, 1990; Hüttenhofer et al, 1996). The eukaryal SECIS structure consists of an elongated stem loop (Martin and Berry, 2001; Korotkov et al, 2002) with an internal loop and non-Watson–Crick quartet tandem (G.A/A.G), which may form a kink-turn motif (Walczak et al, 1996, 1998; Allmang et al, 2002). In addition to this specialized cis-acting element, at least two trans-acting factors, the SECIS binding protein 2 (SBP2) (Copeland et al, 2000, 2001; Low et al, 2000), and a specialized elongation factor (mSelB) (Fagegaltier et al, 2000; Tujebajeva et al, 2000a) are required to achieve redefinition of the UGA codon to selenocysteine by tRNASec decoding. The bacterial elongation factor SelB and associated tRNASec, in contrast, bind directly to the bSECIS located directly adjacent to the stop codon. This implies an important mechanistic distinction, as bSECIS elements act locally at the site of UGA decoding, whereas the positioning of the eukaryal and archaeal SECIS elements to the 3′UTR allows for selenocysteine insertion at one or more UGA codons within the mRNA. Despite the identification of several key components of selenocysteine incorporation, the mechanism by which a ribosome is 'informed' by the 3′UTR SECIS element to direct the appropriate level of selenocysteine incorporation remains unknown. Measurements of selenocysteine incorporation efficiency in bacteria (Suppmann et al, 1999) and in eukaryaea using transfected cells or partially purified translation systems show relatively low-level selenocysteine insertion (Berry et al, 1994; Kollmus et al, 1996; Mehta et al, 2004). Paradoxically, the PHGPx selenoprotein is made at particularly high levels in testis (Ursini et al, 1999), and expression of the selenoprotein P gene, containing 10–17 UGA codons depending upon the species (Hill et al, 1991; Tujebajeva et al, 2000b), would appear to demand efficient incorporation. Incorporation efficiency for specific selenocysteine-encoding mRNAs may be influenced by tissue-specific or other general accessory factors, additional cis-acting elements either within or outside the coding region, or even subcellular localization—complicating measurements of redefinition efficiency in experimental systems. It seems certain that additional cis- and trans-acting factors remain to be identified that modulate the interaction between translational termination and selenocysteine insertion at specific UGA codons to affect redefinition efficiency and regulation in vivo. In the absence of SECIS elements, stop codon redefinition by directed insertion of standard amino acids is commonly achieved by readthrough stimulators located adjacent to the stop codon (Gesteland and Atkins, 1996; Namy et al, 2004). Although it has been suggested that only the single base 3′ of a stop codon can be sufficient to direct programmed readthrough (Li and Rice, 1993), more commonly up to six nucleotides downstream of the redefined codon are involved. Two surveys of the stop codon sequence contexts from nearly 100 known viral examples of readthrough illustrated that a limited number of variants of a 3′ adjacent readthrough motif, CARYYA (Skuzeski et al, 1991), are utilized (Beier and Grimm, 2001; Harrell et al, 2002). In addition, H-type RNA pseudoknot structures (ten Dam et al, 1990) have been shown to direct readthrough in several mammalian retroviruses (Wills et al, 1991; Feng et al, 1992). A well-studied example is gag-pol expression in the murine leukemia virus (MuLV) where the gag UAG stop codon is redefined with approximately 5–10% efficiency (Philipson et al, 1978; Yoshinaka et al, 1985). Both the sequence identity of the eight nucleotides 3′ of the UAG codon (Wills et al, 1994) and key features of the pseudoknot are required activators of readthrough (Alam et al, 1999). Recently, MuLV readthrough levels were shown to be dynamically regulated by binding of the Pol product of MuLV to the eukaryal translational release factor 1 (Orlova et al, 2003). Enhanced readthrough levels attained by this interaction were shown to be important for viral replication. Another example of regulatory stop codon redefinition comes from studies of kelch expression during Drosophila development (Robinson and Cooley, 1997). In this study, the ratio of the termination to readthrough product was suggested to be regulated in a tissue-specific manner. These findings illustrate that not only can redefinition levels be specified by local sequence context for proper gene expression, but also, in some cases, readthrough efficiency is dynamically regulated to achieve optimal gene expression. The occurrence of stop codon readthrough stimulators located adjacent to stop codons, which are redefined as 'standard' amino acids (most often glutamine for UAG and tryptophan for UGA), and selenocysteine-encoding codons in bacteria (with 3′ adjacent bSECIS elements) prompted a re-examination of local eukaryal selenocysteine codon sequence contexts for potential effectors of redefinition. The local sequence contexts of eukaryal selenocysteine-encoding UGA codons were examined for the occurrence of conserved downstream RNA secondary structures. Here we present comparative sequence analysis and experimental data, which supports the existence of a phylogenetically conserved stop codon redefinition element located adjacent to the SEPN1 selenocysteine-encoding UGA codon. Although the biological function of SEPN1 is unknown, mutations in SEPN1 are associated with several early-onset myopathies including rigid spinal muscular dystrophy, classical multiminicore disease, and Mallory body-like form of desmin-related myopathies (Moghadaszadeh et al, 2001; Ferreiro et al, 2002, 2004). Results RNA secondary structure predictions and comparative sequence analysis Sequences located downstream of 36 selenocysteine-encoding UGA codons from 25 human selenoprotein genes, including the selenoprotein P gene, which contains 10 selenocysteine-encoding UGA codons, were examined for potential RNA secondary structures using mfold version 3. 1 (for specific folding parameters and accession numbers, see Materials and methods). Mfold predicts the minimum free energy, ΔG, for multiple RNA foldings of an input RNA sequence (Mathews et al, 1999; Zuker, 2003). The 60 nucleotides located just 3′ of the UGA codon were used as input and then ranked according to the most favorable predicted ΔG value for each RNA sequence. For the 36 selenocysteine insertion sites analyzed, ΔG values for the best RNA folding varied from −2.2 to −36.4 kcal/mol. RNA structures with ΔG values lower than −25 kcal/mol were predicted downstream of five UGA codons; SelO ΔG=−25.8, SEPN1 UGA1 ΔG=−30.5, SEPN1 UGA2 ΔG=−29.5, SelH ΔG=−32.4, and Sps2 ΔG=−36.4 (see Discussion). Analysis of sequences downstream of the 10 selenocysteine-encoding UGA codons in the human selenoprotein P gene reveals a range of ΔG values from −2.2 to −16.6. The identification of several selenocysteine-encoding UGA codons with little potential to form downstream RNA secondary structure suggests that, unlike the case in bacteria, a stem loop near the UGA codon is not required for selenocysteine insertion in eukarya. However, the possibility of structures formed by long-range RNA interactions cannot be ruled out. Based on promising phylogenetic conservation and the identification of sequence variations that maintain base-pairing potential (see below), the sequence surrounding the selenocysteine-encoding codon located in exon 10 of the SEPN1 gene, SEPN1 UGA2, was selected for further examination in this study. SEPN1 genes were identified by BLAST analysis of publicly available protein and nucleotide databases using the human SEPN1 protein sequence as query. Genes with significant similarity to the human SEPN1 gene were identified in 14 vertebrates and two urochordates. Additional BLAST analysis using the distantly related sequence from Ciona intestinalis as query did not identify additional genes with significant similarity to SEPN1. Sequence alignments were performed using ClustalW (Thompson et al, 1994) (Figure 1), revealing a high degree of conservation surrounding the selenocysteine-encoding UGA2 codon. The first UGA codon, UGA1, in the human SEPN1 gene is not phylogenetically conserved and occurs only in an alternately processed transcript with uncertain biological significance. The codon preceding the UGA2 codon and five nucleotides downstream are universally conserved in all SEPN1 genes identified. The potential for a stem loop secondary structure starting seven nucleotides downstream of the UGA codon is predicted and supported by sequence variations that maintain base-pairing potential (Figure 2). The first five base pairs and the size of the predicted stems (17 nucleotides) are preserved. The length of each stem assumes that A:U and G:U pairs are formed at the apical end. These pairings may be nonexistent or only transiently formed in vivo. In addition, three nucleotides in the loop are identical for all predicted structures and a conserved C–A bulge is found after the ninth base pair in the stem of seven out of 13 predicted structures. Figure 1.Sequence alignment of SEPN1 UGA sequence context. The sequence of the selenocysteine insertion site and 64 nucleotides upstream and 101 nucleotides downstream of 16 SEPN1 genes are shown. The location of the UGA codon (X), spacer, and the 5′ (Stem-5′) and 3′ (Stem-3′) strands of the predicted stem loop are indicated above the middle panel of sequences. The UGA codon and nucleotides predicted to be base paired are highlighted in gray. Asterisks indicate positions that are 100% conserved. Download figure Download PowerPoint Figure 2.Secondary structures predicted downstream of SEPN1 UGA codons. The predicted stem loop structures from the 16 SEPN1 genes described in Figure 1 are shown in a two-dimensional representation. Light gray shading is used to indicate codons and black shading with white letters denotes sequence variations that maintain base-pairing potential. Download figure Download PowerPoint An important stem loop The effect of the surrounding sequence context on decoding of the UGA2 codon located in the human SEPN1 gene was examined using a dual luciferase reporter assay in cultured human embryonic kidney cell line (HEK293). A total of 35 nucleotides located 5′ and 46 nucleotides 3′ of the UGA codon were cloned between the Renilla and firefly luciferase reporter genes in plasmid p2luc (Grentzmann et al, 1998) to create the reporter construct UGA2. The firefly luciferase gene lacks an initiation codon and can only be expressed as a fusion protein with Renilla luciferase if translational stop codon readthrough occurs. Readthrough efficiency is calculated as a ratio of the firefly to Renilla luciferase activities standardized to an in-frame control in which the intervening stop codon has been altered to a sense codon (see Materials and methods; Grentzmann et al, 1998). Readthrough efficiency was calculated to be 6% for UGA2 (Figure 3B). An equivalent sequence context was tested for all 10 of the human selenoprotein P UGA codons. Translational readthrough levels were measured at 1% or less for all stop codons with the exception of the first UGA codon from the selenoprotein P gene, which revealed translational readthrough levels of approximately 1.7% (data not shown). The importance of the predicted stem loop in SEPN1 UGA2 for stimulation of translational readthrough was addressed by systematic mutagenesis of the stem loop region. Three nucleotides at a time were changed beginning at the bottom of the stem to interrupt base pairing (Figure 3A). Each disruption mutation reduced stop codon readthrough levels to less than 1% (Figure 3B). Pairing potential was restored at each position by making compensatory mutations such that for each consecutive block of three base pairs in the stem, G:C, A:U, and G:U pairs were altered to C:G, U:A, and U:G, respectively (Figure 3A, SC1-3, SC2-3, SC3-3, SC4-3, SC5-3). Altering the first three base pairs from G:C to C:G (SC1-3) failed to restore readthrough levels. However, restoring base–pairing potential at the second (SC2-3), third (SC3-3), fourth (SC4-3), and fifth (SC5-3) set of three base pairs resulted in partial restoration of stop codon readthrough levels to 5, 1, 2.5, and 4.5%, respectively (Figure 3B). Figure 3.Mutagenic analysis of the human SEPN1 stem loop. The human SEPN1 UGA codon located in exon 10 and surrounding sequence context was cloned into a dual luciferase reporter vector, p2luc, to produce the wild-type construct UGA2. Selective mutations were made in the stem loop region and each construct was expressed in human HEK293 cells and percent readthrough determined. (A) The sequence of the wild-type (UGA2) stem loop region is shown, with base-paired regions highlighted in dark gray (see Supplementary data for complete SEPN1 sequence analyzed). Each subsequent sequence shown corresponds to a unique construct that is identical with the exception of the region highlighted in light gray. (B) Histogram of the calculated percent readthrough for each dual luciferase construct shown in panel A. Error bars indicate standard deviation from the mean. (C) Histogram of the percent readthrough for the dual luciferase constructs containing the UGA2, SC2-1, SC2-2, and SC2-3 sequences in the presence (SECIS) or absence of the SEPN1 3′UTR SECIS element. Download figure Download PowerPoint The stem loop structure predicted for the human SEPN1 gene contains a single C:A mismatch following the ninth base pair of the stem. Converting the C:A mismatch to a C:G base pair increased readthrough levels to 10% (Figure 3B, PS), whereas inverting the C:A mismatch to an A:C mismatch reduced readthrough levels to less than 1% (Figure 3B, IMM). Finally, changing the sequence of the loop reduced readthrough levels to background (Figure 3B, LCa), implying that, in addition to the stem, sequences contained in the loop are important for readthrough stimulation. To determine the ability of this stem loop readthrough element to induce stop codon readthrough in the presence of the natural SEPN1 3′UTR SECIS element, the human SEPN1 SECIS element was cloned into the 3′UTR of the dual luciferase vectors containing the UGA2 stem loop element, or the stem sequence variants SC2-1, SC2-2, and SC2-3 (see Materials and methods). UGA2 readthrough levels increased to approximately 12% with the addition of the SECIS element to the 3′UTR (Figure 3C, UGA2 SECIS). Mutations that disrupt the stem, thereby inactivating the readthrough activity of the stem loop structure (SC2-1 and SC2-2), reduced frameshifting levels to approximately 6% when tested in the presence of the SECIS (Figure 3C, SC2-1 SECIS, SC2-2 SECIS). Restoring the base-pairing potential (SC-3) revealed levels of readthrough (11%) approximating those observed for the wild-type UGA2 in the presence of the SECIS structure (Figure 3C, SC-3 SECIS). These results demonstrate that, in this reporter system, the SEPN1 SECIS element and the stem loop structure each contribute approximately equally to readthrough efficiency at the UGA2 selenocysteine-encoding codon. Stop codon and upstream sequences The ability of the SEPN1 sequences to induce stop codon readthrough of UAA and UAG stop codons was examined in HEK293 cells as described above. When the UGA2 stop codon was replaced with UAG, only a slight reduction in readthrough to 4.5% was observed (Figure 4B, UAG). However, low-level readthrough (<1%) was obtained when UGA was replaced with the UAA stop codon (Figure 4B, UAA). Figure 4.Mutagenic analysis of the human SEPN1 UGA codon and upstream flanking sequence. The human SEPN1 UGA codon located in exon 10 and surrounding sequence context was cloned into a dual luciferase reporter vector, p2luc, to produce the wild-type construct UGA2. The UGA codon was changed to UAG and UAA to test readthrough efficiency for each stop codon and selective mutations were made in the upstream region. Each construct was expressed in human HEK293 cells and percent readthrough determined. (A) The relevant sequence of the wild-type (UGA2) stem loop region is shown. Each subsequent sequence shown corresponds to a unique construct that is identical with the exception of the region highlighted in light gray. (B) Histogram of the calculated percent readthrough for each dual luciferase construct. Error bars indicate standard deviation from the mean. Download figure Download PowerPoint The contribution of SEPN1 upstream RNA sequence to stop codon readthrough efficiency was examined by altering the third position of each codon (with the exception of the single Trp codon, which is encoded by a single codon, UGG) such that the amino-acid sequence encoded was maintained (Figure 4A, 5′ 3POS). Stop codon efficiency was reduced approximately two-fold by these changes (Figure 4B, 5′ 3POS). In a second construct, a single base was deleted from the 5′ end of the readthrough cassette and a single base was inserted three nucleotides prior to the stop codon (Figure 4A, 5′ FS). In this case, the RNA sequence is maintained essentially intact but the sequence of the nascent peptide is changed due to the shift in reading frame. Changing the composition of the nascent peptide did not significantly alter readthrough efficiency. A significant spacer sequence The RNA sequences located between the stop codon and the stem loop were changed to examine the effect of this spacer region on stop codon readthrough efficiency (Figure 5A). Mutagenesis of the six-nucleotide spacer, GGUUCA, was tested using the dual luciferase reporter system in HEK293 cells as described above. Comparison of the six-nucleotide SEPN1 spacer region GGUUCA to the previously identified CARYYA readthrough motif revealed a match for the last three nucleotides. Altering the last three nucleotides to AGU or ACA resulted in a modest, approximately two-fold, decrease in readthrough efficiency (Figure 5B, Spa, Spd). However, alterations that retain the YYA composition at this site, CUA or UUA, maintained readthrough levels near wild-type efficiency (Figure 5B, Spb, Spc). When the first three nucleotides of the spacer were changed to CAG and CAA to match the CARYYA motif, readthrough efficiency increased to 12 and 17%, respectively (Figure 5B, Spe, Spf). The contribution of the first three nucleotides of the spacer to readthrough efficiency was further examined by changing each nucleotide independently to a C (Figure 5A, Spg, Sph, Spi). Changing either of the first two nucleotides to a C reduced readthrough levels to 0.2 and 3%, respectively (Figure 5B, Spg, Sph), whereas changing the third nucleotide had no appreciable effect on readthrough (Figure 5B, Spi). The role of spacer length was examined by changing the length to eight nucleotides (Figure 5A, Spj, Spk, Spl) or four nucleotides (Figure 5A, Spm). Each resulted in a reduction in readthrough efficiency to approximately 2% (Figure 5B). Figure 5.Mutagenic analysis of the human SEPN1 spacer sequence. Selective mutations were made to the spacer sequence in the wild-type construct UGA2 and each construct was expressed in human HEK293 cells and percent readthrough determined. (A) The relevant sequence of the wild-type (UGA2) stem loop region is shown, with base-paired regions highlighted in dark gray. Each subsequent sequence shown corresponds to a unique construct that is identical with the exception of the region highlighted in light gray. (B) Histogram of the calculated percent readthrough for each dual luciferase construct shown in panel A. Error bars indicate standard deviation from the mean. Download figure Download PowerPoint The RNA element described here contains a key sequence separating the UGA codon from the essential downstream stem loop structure. Mutations designed to conform the spacer to the previously identified CARYYA motif (Skuzeski et al, 1991; Beier and Grimm, 2001; Harrell et al, 2002) resulted in higher readthrough levels, with the exception of changing the first G of the SEPN1 spacer to C, which eliminated readthrough. Those changes that were designed to reduce similarity to the known readthrough motif lowered readthrough efficiency. The spacer length is also critical and likely serves to position the structure at the predicted distance to be near the mRNA entrance site (Yusupova et al, 2001) and the ribosome mRNA unwinding center ('helicase') (Takyar et al, 2005). MuLV gag stop codon readthrough stimulation by the SEPN1 stem loop Comparison of the spacer sequence of SEPN1 to that of the spacer region known to be important for MuLV readthrough reveals that the first two and last three nucleotides are identical; MuLV spacer=GGAGGUCA (Figure 5). A dual luciferase reporter was constructed that contains the MuLV stop codon (UAG) and downstream pseudoknot (Figure 6A, MuLVWT). The ability of the SEPN1 stem loop to stimulate readthrough of the MuLV stop codon was tested by replacing the MuLV pseudoknot with the SEPN1 stem loop (Figure 6A, MuLV1). Readthrough efficiency promoted by the wild-type MuLV pseudoknot was 7% and replacing the pseudoknot with the SEPN1 stem loop reduced readthrough to 1% (Figure 6B, MuLVwt, MuLV1). Reducing the spacer length to six nucleotides by replacing it with the SEPN1 spacer or deleting the first and fourth Gs of the MuLV spacer partially restored readthrough efficiency to approximately 3% (Figure 6B, MuLV2, MuLV3). Adding two nucleotides to the end or between the GGU and UCA of the SEPN1 spacer reduced readthrough of the UAG to 1 and 0.5% respectively (Figure 6B, MuLV4, MuLV7), and 2% readthrough was observed with an eight-nucleotide spacer, GGGUGUCA (Figure 6B, MuLV8). Finally, the MuLV and SEPN1 spacer regions do not induce high-level readthrough in the absence of downstream structures, as readthrough efficiency dropped to approximately 0.3% when the MuLV or SEPN1 structures were deleted entirely (Figure 6B, MuLV5, MuLV6). Figure 6.Comparison of MuLV and SEPN1 readthrough stimulators. The MuLV readthrough sequences were cloned into a dual luciferase reporter vector, p2luc, to produce the MuLVWT construct. The MuLV pseudoknot was replaced by the SEPN1 stem loop and selective mutations were made to the spacer sequences and each construct was expressed in human HEK293 cells by transient transfection and percent readthrough determined. (A) The sequence of the MuLV readthrough region (MuLVWT) is shown with the stop codon in light gray, and regions of each stem of the readthrough pseudoknot that are predicted to be base paired are shown highlighted in dark gray (Stem I) and light gray (Stem II). The MuLV pseudoknot was deleted for MuLV5/6, and replaced by the SEPN1 stem loop for MuLV1, 2, 3, 4, 7, and 8. Each subsequent sequence shown corresponds to a unique construct, with changes to the spacer region highlighted in light gray. (B) Histogram of the calculated percent readthrough for each dual luciferase construct shown in panel A. Error bars indicate standard deviation from the mean. Download figure Download PowerPoint The strong sequence bias for the six nucleotides located downstream of redefined stop codons, experimental evidence that the spacer region in MuLV contains key sequence stimulators of gag-pol readthrough (Wills et al, 1994), and mutagenesis of the SEPN1 spacer region further illustrate that sequences located adjacent to stop codons can be utilized as a means to facilitate stop codon readthrough for gene expression purposes. In addition, structure 'swapping' experiments here demonstrate that the SEPN1 stem loop can induce stop codon readthrough of the MuLV gag stop codon, albeit at a lower level than the wild-type MuLV pseudoknot structure. Discussion Phylogene
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