Selenocysteine insertion sequence binding protein 2L is implicated as a novel post-transcriptional regulator of selenoprotein expression

The amino acid selenocysteine (Sec) is encoded by UGA codons. Recoding of UGA from stop to Sec requires a Sec insertion sequence (SECIS) element in the 3' UTR of selenoprotein mRNAs. SECIS binding protein 2 (SBP2) binds the SECIS element and is essential for Sec incorporation into the nascent peptide. SBP2-like (SBP2L) is a paralogue of SBP2 in vertebrates and is the only SECIS binding protein in some invertebrates where it likely directs Sec incorporation. However, vertebrate SBP2L does not promote Sec incorporation in in vitro assays. Here we present a comparative analysis of SBP2 and SBP2L SECIS binding properties and demonstrate that its inability to promote Sec incorporation is not due to lower SECIS affinity but likely due to lack of a SECIS dependent domain association that is found in SBP2. Interestingly, however, we find that an invertebrate version of SBP2L is fully competent for Sec incorporation in vitro. Additionally, we present the first evidence that SBP2L interacts with selenoprotein mRNAs in mammalian cells, thereby implying a role in selenoprotein expression.     

Lack of secondary structure characterizes the 5' ends of mammalian mitochondrial mRNAs

The mammalian mitochondrial genome encodes 13 proteins, which are synthesized at the direction of nine monocistronic and two dicistronic mRNAs. These mRNAs lack both 5' and 3' untranslated regions. The mechanism by which the specialized mitochondrial translational apparatus locates start codons and initiates translation of these leaderless mRNAs is currently unknown. To better understand this mechanism, the secondary structures near the start codons of all 13 open reading frames have been analyzed using RNA SHAPE chemistry. The extent of structure in these mRNAs as assessed experimentally is distinctly lower than would be predicted by current algorithms based on free energy minimization alone. We find that the 5' ends of all mitochondrial mRNAs are highly unstructured. The first 35 nucleotides for all mitochondrial mRNAs form structures with free energies less favorable than -3 kcal/mol, equal to or less than a single typical base pair. The start codons, which lie at the very 5' ends of these mRNAs, are accessible within single stranded motifs in all cases, making them potentially poised for ribosome binding. These data are consistent with a model in which the specialized mitochondrial ribosome preferentially allows passage of unstructured 5' sequences into the mRNA entrance site to participate in translation initiation.     

Selenocysteine incorporation in eukaryotes: insights into mechanism and efficiency from sequence, structure, and spacing proximity studies of the type 1 deiodinase SECIS element.

SECIS elements are stem-loop structures located in the 3' untranslated regions (UTRs) of eukaryotic selenoprotein mRNAs that are required for directing cotranslational selenocysteine incorporation at UGA codons. In prokaryotes, stem-loops mediating selenocysteine incorporation are located immediately downstream of the UGA selenocysteine codon, in the coding region. Previous characterization studies of the mammalian SECIS elements of type 1 deiodinase, glutathione peroxidase, and selenoprotein P showed that conserved nucleotides in the loops and unpaired bulges, and base pairing in the stems are required for SECIS function. These initial studies utilized approximately 175-230-nt segments of the 3'UTRs of the selenoprotein mRNAs. Here we define the minimal functional rat type 1 deiodinase SECIS element, a 45-nt segment, the 5' boundary of which corresponds precisely to the 5'-most critical conserved nucleotide identified previously. We also define base pairing requirements in the stem of this element. In view of the presence of SECIS elements in the open reading frames (ORFs) of bacterial selenoproteins, we examine the effects in the type 1 deiodinase of extending the ORF into the SECIS element, and find that this dramatically inhibits SECIS function. Finally, we define a minimal spacing requirement of 51-111 nt between a eukaryotic UGA selenocysteine codon and SECIS element.     

cDNA cloning,expression pattern and RNA binding analysis of human selenocysteine insertion sequence (SECIS) binding protein 2

Selenocysteine and selenoprotein synthesis require a complex molecular machinery in mammals. Among the key players is the RNA-protein complex formed by the selenocysteine insertion sequence (SECIS) binding protein (SBP2) and the SECIS element, an RNA hairpin in the 3' untranslated regions of selenoprotein messenger RNAs (mRNAs). We have isolated the DNA complementary to mRNA of the human SBP2, enabling us to establish that it differs from a previously reported human SBP2-like protein. Examination of the expression pattern revealed that the human SBP2 protein is encoded by a 4 kb long mRNA that is over-expressed in testis. Compared to the rat SBP2 sequence, the human SBP2 protein displays two highly conserved domains with 92 and 95% amino acid identity, the latter one containing the RNA binding domain. The inter-domain section carries 55% sequence identity, the remainder of the SBP2 sequences showing about 65% identity, values lower than expected for two mammalian proteins. Interestingly, we could show that the binding of human SBP2 to the SECIS RNA is stimulated by the selenoprotein-specialized elongation translation factor mSelB/eEFsec.     

Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons.

We investigated the requirements for selenocysteine insertion at single or multiple UGA codons in eukaryotic selenoproteins. Two functional SECIS elements were identified in the 3' untranslated region of the rat selenoprotein P mRNA, with predicted stem-loops and critical nucleotides similar to those in the SECIS elements in the type I iodothyronine 5' deiodinase (5'DI) and glutathione peroxidase selenoprotein mRNAs. Site-directed mutational analyses of three SECIS elements confirmed that conserved nucleotides in the loop and in unpaired regions of the stem are critical for activity. This indicates that multiple contact sites are required for SECIS function. Stop codon function at any of five out-of-context UGA codons in the 5'DI mRNA was suppressed by SECIS elements from the 5'DI or selenoprotein P genes linked downstream. Thus, the presence of SECIS elements in eukaryotic selenoprotein mRNAs permits complete flexibility in UGA codon position.     

哺乳动物硒蛋白的研究进展

硒是哺乳动物和人必需的微是元素。硒的生物学功能主要是以硒蛋白的形式表现的。到目前为止,已经克隆并测定ｃＤＮＡ顺序的哺乳动物硒蛋白有９种停,它们是细胞内谷胱甘肽过氧化物酶、细胞外谷胱甘肽过氧化物酶、磷脂氢谷胱甘肽过氧化物酶、胃肠谷胱甘肽过氧化物酶、Ｉ型碘化甲状腺原氨酸５′脱碘酶、Ⅱ型碘化甲状腺原氨酸５′脱磺酶、Ⅲ型碘化甲状腺原氨酸５′脱碘酶、硒蛋白Ｐ和硒蛋白Ｗ。这些硒蛋白中硒参入到蛋白分子是通过硒半     

Truncated forms of the dual function human ASCT2 neutral amino acid transporter/retroviral receptor are translationally initiated at multiple alternative CUG and GUG codons

The sodium-dependent neutral amino acid transporter type 2 (ASCT2) was recently identified as a cell surface receptor for endogenously inherited retroviruses of cats, baboons, and humans as well as for horizontally transmitted type-D simian retroviruses. By functional cloning, we obtained 10 full-length 2.9-kilobase pair (kbp) cDNAs and two smaller identical 2.1-kbp cDNAs that conferred susceptibility to these viruses. Compared with the 2.9-kbp cDNA, the 2.1-kbp cDNA contains exonic deletions in its 3' noncoding region and a 627-bp 5' truncation that eliminates sequences encoding the amino-terminal portion of the full-length ASCT2 protein. Although expression of the truncated mRNA caused enhanced amino acid transport and viral receptor activities, the AUG codon nearest to its 5' end is flanked by nucleotides that are incompatible with translational initiation and the next in-frame AUG codon is far downstream toward the end of the protein coding sequence. Interestingly, the 5' region of the truncated ASCT2 mRNA contains a closely linked series of CUG(Leu) and GUG(Val) codons in optimal consensus contexts for translational initiation. By deletion and site-directed mutagenesis, cell-free translation, and analyses of epitope-tagged ASCT2 proteins synthesized intracellularly, we determined that the truncated mRNA encodes multiple ASCT2 isoforms with distinct amino termini that are translationally initiated by a leaky scanning mechanism at these CUG and GUG codons. Although the full-length ASCT2 mRNA contains a 5'-situated AUG initiation codon, a significant degree of leaky scanning also occurred in its translation. ASCT2 isoforms with relatively short truncations were active in both amino acid transport and viral reception, whereas an isoform with a 79-amino acid truncation that lacked the first transmembrane sequence was active only in viral reception. We conclude that ASCT2 isoforms with truncated amino termini are synthesized in mammalian cells by a leaky scanning mechanism that employs multiple alternative CUG and GUG initiation codons.     

Coupled tRNA(Sec)-dependent assembly of the selenocysteine decoding apparatus

SECIS elements recode UGA codons from "stop" to "sense." These RNA secondary structures, present in eukaryotic selenoprotein mRNA 3' 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.     

Mammalian selenoprotein in which selenocysteine (Sec) incorporation is supported by a new form of Sec insertion sequence element

Selenocysteine (Sec), the 21st amino acid in protein, is encoded by UGA. The Sec insertion sequence (SECIS) element, which is the stem-loop structure present in 3' untranslated regions (UTRs) of eukaryotic selenoprotein-encoding genes, is essential for recognition of UGA as a codon for Sec rather than as a stop signal. We now report the identification of a new eukaryotic selenoprotein, designated selenoprotein M (SelM). The 3-kb human SelM-encoding gene has five exons and is located on chromosome 22 but has not been correctly identified by either Celera or the public Human Genome Project. We characterized human and mouse SelM cDNA sequences and expressed the selenoprotein in various mammalian cell lines. The 3" UTR of the human, mouse, and rat SelM-encoding genes lacks a canonical SECIS element. Instead, Sec is incorporated in response to a conserved mRNA structure, in which cytidines are present in place of the adenosines previously considered invariant. Substitution of adenosines for cytidines did not alter Sec incorporation; however, other mutant structures did not support selenoprotein synthesis, demonstrating that this new form of SECIS element is functional. SelM is expressed in a variety of tissues, with increased levels in the brain. It is localized to the perinuclear structures, and its N-terminal signal peptide is necessary for protein translocation.     

Cloning and characterization of zRICH, a 2',3'-cyclic-nucleotide 3'-phosphodiesterase induced during zebrafish optic nerve regeneration

We previously reported cloning of cDNAs encoding both components of a protein doublet induced during goldfish optic nerve regeneration. The predicted protein sequences showed significant homology with the mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases (CNPases). CNPases are well-established markers of mammalian myelin; hence, the cDNAs were designated gRICH68 and gRICH70 (for goldfish Regeneration-Induced CNPase Homologues of 68 and 70 kDa). Homologous cDNAs have now been isolated from zebrafish encoding a highly related protein, which we have termed zRICH. RNase protection assays show that zRICH mRNA is induced significantly (fivefold) in optic nerve regenerating zebrafish retinas 7 days following nerve crush. Western blots show a single band in zebrafish brain and retina extracts, with immunoreactivity increasing three-fold in regenerating retinas 21 days postcrush. Immunohistochemical analysis indicated that this increase in zRICH protein expression is localized to the retinal ganglion cell layer in regenerating retina. We have characterized and evaluated the relevance of a conserved beta-ketoacyl synthase motif in zRICH to CNPase activity by means of site-directed mutagenesis. Two residues within the motif, H334 and T336, are critical for enzymatic activity. A cysteine residue within the motif, which corresponds to a critical residue for beta-ketoacyl synthase, does not appear to participate in the phosphodiesterase activity.     

A protein binds the selenocysteine insertion element in the 3'-UTR of mammalian selenoprotein mRNAs.

Several gene products are involved in co-translational insertion of selenocysteine by the tRNA(Sec). In addition, a stem-loop structure in the mRNAs coding for selenoproteins is essential to mediate the selection of the proper selenocysteine UGA codon. Interestingly, in eukaryotic selenoprotein mRNAs, this stem-loop structure, the selenocysteine insertion sequence (SECIS) element, resides in the 3'-untranslated region, far downstream of the UGA codon. In view of unravelling the underlying complex mechanism, we have attempted to detect RNA-binding proteins with specificity for the SECIS element. Using mobility shift assays, we could show that a protein, present in different types of mammalian cell extracts, possesses the capacity of binding the SECIS element of the selenoprotein glutathione peroxidase (GPx) mRNA. We have termed this protein SBP, for Secis Binding Protein. Competition experiments attested that the binding is highly specific and UV cross-linking indicated that the protein has an apparent molecular weight in the range of 60-65 kDa. Finally, some data suggest that the SECIS elements in the mRNAs of GPx and another selenoprotein, type I iodothyronine 5' deiodinase, recognize the same SBP protein. This constitutes the first report of the existence of a 3' UTR binding protein possibly involved in the eukaryotic selenocysteine insertion mechanism.     

An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine.

In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3' UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3' UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.     

A developmental study of rat sperm and testis selenoproteins

Essentially all of the selenium in the rat spermatozoon is bound to a polypeptide of Mr 15,000-17,000 confined to the capsule that surrounds the sperm mitochondria. Isoelectric focussing of isolated 75Se-labelled, carboxymethylated mitochondrial capsule protein (MCP) reveals the presence of at least four radioactive components, with a predominant charge isomer at pI4.6. The sperm selenoprotein appears to be identical with MCP, as judged by the exact coincidence of radioactivity and protein stain during two-dimensional electrophoresis. The temporal pattern of 75Se-labelling of rat caput epididymal spermatozoa after intratesticular 75Se injection suggests that maximum incorporation of 75Se into MCP occurs in step 7-step 12 spermatids and that 75Se uptake ceases during step 15 of spermiogenesis. The developmental appearance of sperm selenoprotein in rat testis therefore appears to lag several days behind that reported for MCP in mouse testis, suggesting the presence of selenium-free MCP in immature germ cells. SDS gel electrophoretic analysis of testis subcellular fractions 24 h after 75Se injection into rat testis at 21, 28 and 90 days of age indicates that sperm selenoprotein first appears in very low concentration during late meiosis and that its concentration increases sharply during early spermiogenesis. Additional 75Se-labelled polypeptides were detected on the gels, most of them of higher molecular weight than MCP. At least two of these (Mr 47,000 and 54,000) displayed a marked decrease in labelling between 5 and 24 h after injection into adult testis, coincident with a comparable increase in 75Se-labelled MCP, indicating that they may be precursors of MCP.     

The cDNA for rat selenoprotein P contains 10 TGA codons in the open reading frame

Selenoprotein P is a plasma protein recently purified and characterized as containing 7.5 +/- 1.0 selenium atoms/molecule as selenocysteine. In rats maintained on a defined diet containing nutritionally adequate amounts of selenate as the sole selenium source, over half the selenium in plasma is accounted for by selenoprotein P. Its cDNA has been cloned from a rat liver library and sequenced. The sequence is highly unusual, containing 10 TGA codons in its open reading frame prior to the TAA termination codon. TGA designates selenocysteine in other selenoproteins, and limited peptide sequencing that included the amino acids encoded by two of the TGA codons verified that they correspond to selenocysteine. The deduced 366-amino acid sequence is histidine- and cysteine-rich and contains 9 of its selenocysteines in the terminal 122 amino acids. Comparison of the deduced amino acid sequence of selenoprotein P with those of other selenoprotein reveals no significant similarities. Selenoprotein P represents a new class of selenoproteins and is the first protein described with more than 1 selenocysteine in a single polypeptide chain. The primary structure of selenoprotein P suggests that it might be responsible for some of the antioxidant properties of selenium.     

Functional interaction of phospholipid hydroperoxide glutathione peroxidase with sperm mitochondrion-associated cysteine-rich protein discloses the adjacent cysteine motif as a new substrate of the selenoperoxidase

The mitochondrial capsule is a selenium- and disulfide-rich structure enchasing the outer mitochondrial membrane of mammalian spermatozoa. Among the proteins solubilized from the sperm mitochondrial capsule, we confirmed, by using a proteomic approach, the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPx) as a major component, and we also identified the sperm mitochondrion-associated cysteine-rich protein (SMCP) and fragments/aggregates of specific keratins that previously escaped detection (Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J., and Flohé, L. (1999) Science 285, 1393-1396). The evidence for a functional association between PHGPx, SMCP, and keratins is further supported by the identification of a sequence motif of regularly spaced Cys-Cys doublets common to SMCP and high sulfur keratin-associated proteins, involved in bundling hair shaft keratin by disulfide cross-linking. Following the oxidative polymerization of mitochondrial capsule proteins, catalyzed by PHGPx, two-dimensional redox electrophoresis analysis showed homo- and heteropolymers of SMCP and PHGPx, together with other minor components. Adjacent cysteine residues in SMCP peptides are oxidized to cystine by PHGPx. This unusual disulfide is known to drive, by reshuffling oxidative protein folding. On this basis we propose that oxidative polymerization of the mitochondrial capsule is primed by the formation of cystine on SMCP, followed by reshuffling. Occurrence of reshuffling is further supported by the calculated thermodynamic gain of the process. This study suggests a new mechanism where selenium catalysis drives the cross-linking of structural elements of the cytoskeleton via the oxidation of a keratin-associated protein.     

Two genes encoding the bovine mitochondrial ATP synthase proteolipid specify precursors with different import sequences and are expressed in a tissue-specific manner.

Two cDNAs encoding different precursor proteins of the same mature proteolipid subunit of mitochondrial ATP synthase have been cloned from a bovine cDNA library. The hybridisation probe was a mixture of 17-mer oligonucleotides containing 256 discrete sequences. The coding sequences of the two cDNAs differ in 25 silent positions of codons and the 3' non-coding sequences are only weakly related. The precursor sequences, which direct the import of the proteolipid into the mitochondrion, are 61 and 68 amino acids long. They are related to each other in regions which probably are recognition signals for the processing protease. The corresponding genes are expressed differently in various tissues in a way that reflects their embryonic origin.     

The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria.

Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.