The Experts below are selected from a list of 198 Experts worldwide ranked by ideXlab platform
Donna M Driscoll - One of the best experts on this subject based on the ideXlab platform.
-
identification of nucleotides and amino acids that mediate the interaction between ribosomal protein l30 and the SECIS Element
BMC Molecular Biology, 2013Co-Authors: Abby L Bifano, Donna M Driscoll, Tarik Atassi, Tracey M FerraraAbstract:Ribosomal protein L30 belongs to the L7Ae family of RNA-binding proteins, which recognize diverse targets. L30 binds to kink-turn motifs in the 28S ribosomal RNA, L30 pre-mRNA, and mature L30 mRNA. L30 has a noncanonical function as a component of the UGA recoding machinery that incorporates selenocysteine (Sec) into selenoproteins during translation. L30 binds to a putative kink-turn motif in the Sec Insertion Sequence (SECIS) Element in the 3’ UTR of mammalian selenoprotein mRNAs. The SECIS also interacts with SECIS-binding protein 2 (SBP2), an essential factor for Sec incorporation. Previous studies showed that L30 and SBP2 compete for binding to the SECIS in vitro. The SBP2:SECIS interaction has been characterized but much less is known about how L30 recognizes the SECIS. Here we use enzymatic RNA footprinting to define the L30 binding site on the SECIS. Like SBP2, L30 protects nucleotides in the 5’ side of the internal loop, the 5’ side of the lower helix, and the SECIS core, including the GA tandem base pairs that are predicted to form a kink-turn. However, L30 has additional determinants for binding as it also protects nucleotides in the 3’ side of the internal loop, which are not protected by SBP2. In support of the competitive binding model, we found that purified L30 repressed UGA recoding in an in vitro translation system, and that this inhibition was rescued by SBP2. To define the amino acid requirements for SECIS-binding, site-specific mutations in L30 were generated based on published structural studies of this protein in a complex with its canonical target, the L30 pre-mRNA. We identified point mutations that selectively inhibited binding of L30 to the SECIS, to the L30 pre-mRNA, or both RNAs, suggesting that there are subtle differences in how L30 interacts with the two targets. This study establishes that L30 and SBP2 bind to overlapping but non-identical sites on the SECIS. The amino acid requirements for the interaction of L30 with the SECIS differ from those that mediate binding to the L30 pre-mRNA. Our results provide insight into how L7Ae family members recognize their cognate RNAs.
-
alternative transcripts and 3 utr Elements govern the incorporation of selenocysteine into selenoprotein s
PLOS ONE, 2013Co-Authors: Jodi L Bubenik, Donna M Driscoll, Angela C MiniardAbstract:Selenoprotein S (SelS) is a 189 amino acid trans-membrane protein that plays an important yet undefined role in the unfolded protein response. It has been proposed that SelS may function as a reductase, with the penultimate selenocysteine (Sec188) residue participating in a selenosulfide bond with cysteine (Cys174). Cotranslational incorporation of Sec into SelS depends on the recoding of the UGA codon, which requires a Selenocysteine Insertion Sequence (SECIS) Element in the 3′UTR of the transcript. Here we identify multiple mechanisms that regulate the expression of SelS. The human SelS gene encodes two transcripts (variants 1 and 2), which differ in their 3′UTR sequences due to an alternative splicing event that removes the SECIS Element from the variant 1 transcript. Both transcripts are widely expressed in human cell lines, with the SECIS-containing variant 2 mRNA being more abundant. In vitro experiments demonstrate that the variant 1 3′UTR does not allow readthrough of the UGA/Sec codon. Thus, this transcript would produce a truncated protein that does not contain Sec and cannot make the selenosulfide bond. While the variant 2 3′UTR does support Sec insertion, its activity is weak. Bioinformatic analysis revealed two highly conserved stem-loop structures, one in the proximal part of the variant 2 3′UTR and the other immediately downstream of the SECIS Element. The proximal stem-loop promotes Sec insertion in the native context but not when positioned far from the UGA/Sec codon in a heterologous mRNA. In contrast, the 140 nucleotides downstream of the SECIS Element inhibit Sec insertion. We also show that endogenous SelS is enriched at perinuclear speckles, in addition to its known localization in the endoplasmic reticulum. Our results suggest the expression of endogenous SelS is more complex than previously appreciated, which has implications for past and future studies on the function of this protein.
-
Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression
Nucleic acids research, 2010Co-Authors: Angela C Miniard, Lisa M. Middleton, Michael E. Budiman, Carri A. Gerber, Donna M DriscollAbstract:Selenium, an essential trace Element, is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. In order to synthesize selenoproteins, a translational reprogramming event must occur since Sec is encoded by the UGA stop codon. In mammals, the recoding of UGA as Sec depends on the selenocysteine insertion sequence (SECIS) Element, a stem-loop structure in the 3′ untranslated region of the transcript. The SECIS acts as a platform for RNA-binding proteins, which mediate or regulate the recoding mechanism. Using UV crosslinking, we identified a 110 kDa protein, which binds with high affinity to SECIS Elements from a subset of selenoprotein mRNAs. The crosslinking activity was purified by RNA affinity chromatography and identified as nucleolin by mass spectrometry analysis. In vitro binding assays showed that purified nucleolin discriminates among SECIS Elements in the absence of other factors. Based on siRNA experiments, nucleolin is required for the optimal expression of certain selenoproteins. There was a good correlation between the affinity of nucleolin for a SECIS and its effect on selenoprotein expression. As selenoprotein transcript levels and localization did not change in siRNA-treated cells, our results suggest that nucleolin selectively enhances the expression of a subset of selenoproteins at the translational level.
-
novel structural determinants in human SECIS Elements modulate the translational recoding of uga as selenocysteine
Nucleic Acids Research, 2009Co-Authors: Lynda Latreche, Olivier Jeanjean, Donna M Driscoll, Laurent ChavatteAbstract:The selenocysteine insertion sequence (SECIS) Element directs the translational recoding of UGA as selenocysteine. In eukaryotes, the SECIS is located downstream of the UGA codon in the 3′-UTR of the selenoprotein mRNA. Despite poor sequence conservation, all SECIS Elements form a similar stem-loop structure containing a putative kink-turn motif. We functionally characterized the 26 SECIS Elements encoded in the human genome. Surprisingly, the SECIS Elements displayed a wide range of UGA recoding activities, spanning several 1000-fold in vivo and several 100-fold in vitro. The difference in activity between a representative strong and weak SECIS Element was not explained by differential binding affinity of SECIS binding Protein 2, a limiting factor for selenocysteine incorporation. Using chimeric SECIS molecules, we identified the internal loop and helix 2, which flank the kink-turn motif, as critical determinants of UGA recoding activity. The simultaneous presence of a GC base pair in helix 2 and a U in the 5′-side of the internal loop was a statistically significant predictor of weak recoding activity. Thus, the SECIS contains intrinsic information that modulates selenocysteine incorporation efficiency.
-
insight into mammalian selenocysteine insertion domain structure and ribosome binding properties of sec insertion sequence binding protein 2
Molecular and Cellular Biology, 2001Co-Authors: Paul R Copeland, Vincent Stepanik, Donna M DriscollAbstract:There are many examples of translational regulation that involve RNA binding proteins interacting with specific sequences in the 3′ untranslated regions (UTRs) of various mRNAs (10, 18). A specialized case of this is found in the incorporation of selenocysteine (Sec) into a select group of eukaryotic proteins where a sequence specific 3′ UTR binding protein is required for Sec insertion at its cognate UGA codon (6). The Sec-containing proteins that have been characterized perform myriad biological functions including oxidant defense and hormone maturation (reviewed in references 9 and 20). While mice deficient in the selenoprotein glutathione peroxidase are viable (11), one or more selenoproteins are apparently required for early development, as elimination of the tRNASec gene in mice causes early embryo lethality (3). While the incorporation of Sec into the Escherichia coli formate dehydrogenase isozymes is fairly well characterized (2), the system which governs mammalian Sec insertion has only been partially elucidated. Several essential components of what we here term the Sec insertion complex (SIC) have been identified. These include the UGA codon that encodes Sec (13), the Sec insertion sequence (SECIS) Element found in all selenoprotein 3′ UTRs (1), and the recently identified SECIS binding protein (SBP2), which we demonstrated to be required for Sec insertion in vitro (6). A fourth component of the SIC that is also likely to be required for Sec incorporation is the Sec-specific elongation factor (eEFsec) that has been recently shown to be functional in transfected cells (7, 21). Because Sec is encoded by what is ordinarily a stop codon, the mechanism of Sec insertion is likely to involve direct competition with translation termination. The SIC may therefore interact with or regulate the interactions of the release factors required for translation termination. This work focuses on the RNA binding protein SBP2, a novel 94-kDa protein that shares a 32-amino acid motif with several ribosomal proteins and eukaryotic translation termination release factor 1 (eRF-1). eRF-1 functionally and structurally mimics a tRNA molecule that is specific for stop codons and in this fashion binds the ribosomal A site and terminates chain elongation (19). The fact that SBP2 and eRF-1 share this sequence suggests that they also share a common class of targets, which may shed light on the molecular basis for the competition between Sec insertion and termination. This conserved sequence has been proposed to be a novel RNA binding motif (12) and has recently been demonstrated to be involved in the binding of ribosomal protein L30 to its own mRNA (15). In this report, we establish that this motif, which we will refer to as the L30 RNA binding domain, is required for SBP2 RNA binding activity and function as measured by the ability to incorporate Sec in vitro. In addition, we have identified a nonoverlapping functional domain that is required for Sec insertion but not RNA binding. Further analysis indicates that SBP2 is stably associated with the ribosomal fraction in transfected cells and in vitro, and that this interaction may be mediated by 28S rRNA. We hypothesize that SBP2 may be involved in selecting a subset of ribosomes which are competent for Sec insertion.
Paul R Copeland - One of the best experts on this subject based on the ideXlab platform.
-
the polypyrimidine tract binding protein ptbp1 regulates selenium homeostasis via the selenoprotein p 3 untranslated region
bioRxiv, 2020Co-Authors: Sumangala P Shetty, Nora T Kiledjian, Paul R CopelandAbstract:Selenoproteins contain the 21st amino acid, selenocysteine (Sec), which is incorporated at select UGA codons when the encoding mRNA contains a specialized hairpin sequence in its 39 UTR. This hairpin, the so-called Sec insertion sequence (SECIS) Element, is found in all selenoprotein mRNAs, but the sequence surrounding these Elements is widely variable and in many cases of considerable length. In order to determine the function of one such SECIS context, we chose to focus on the plasma selenoprotein, SELENOP, that is required to maintain selenium homeostasis. It is unique in that its mRNA contains two SECIS Elements that lie in the context of a highly conserved 843-nucleotide 39 UTR. Prior work has attempted to examine the functions of the SECIS context but none were identified. Here we have used CRISPR/Cas9 genome editing to delete the region between the two SECIS Elements. We found that this sequence is required to mediate an increase in SELENOP synthesis under conditions of peroxide stress. Using RNA affinity chromatography, we have identified PTBP1 as the major RNA binding protein that specifically interacts with this region.
-
processive incorporation of multiple selenocysteine residues is driven by a novel feature of the selenocysteine insertion sequence
Journal of Biological Chemistry, 2018Co-Authors: Sumangala P Shetty, Ryan J Sturts, Michael B Vetick, Paul R CopelandAbstract:: RNA stem loop structures have been frequently shown to regulate essential cellular processes. The selenocysteine insertion sequence (SECIS) Element, found in the 3' UTRs of all selenoprotein mRNAs, is an example of such a structure, as it is required for the incorporation of the 21st amino acid, selenocysteine (Sec). Selenoprotein synthesis poses a mechanistic challenge because Sec is incorporated during translation in response to a stop codon (UGA). Although it is known that a SECIS-binding protein (SBP2) is required for Sec insertion, the mechanism of action remains elusive. Additional complexity is present in the synthesis of selenoprotein P (SELENOP), which is the only selenoprotein that contains multiple UGA codons and possesses two SECIS Elements in its 3' UTR. Thus, full-length SELENOP synthesis requires processive Sec incorporation. Using zebrafish Selenop, in vitro translation assays, and 75Se labeling in HEK293 cells, we found here that processive Sec incorporation is an intrinsic property of the SECIS Elements. Specifically, we identified critical features of SECIS Elements that are required for processive Sec incorporation. A screen of the human SECIS Elements revealed that most of these Elements support processive Sec incorporation in vitro; however, we also found that the processivity of Sec incorporation into Selenop in cells is tightly regulated. We propose a model for processive Sec incorporation that involves differential recruitment of SECIS-binding proteins.
-
selenocysteine insertion sequence SECIS binding protein 2 alters conformational dynamics of residues involved in trna accommodation in 80 s ribosomes
Journal of Biological Chemistry, 2012Co-Authors: Kelvin Caban, Paul R CopelandAbstract:Abstract Sec-tRNASec is site-specifically delivered at defined UGA codons in selenoprotein mRNAs. This recoding event is specified by the selenocysteine insertion sequence (SECIS) Element and requires the selenocysteine (Sec)-specific elongation factor, eEFSec, and the SECIS binding protein, SBP2. Sec-tRNASec is delivered to the ribosome by eEFSec-GTP, but this ternary complex is not sufficient for Sec incorporation, indicating that its access to the ribosomal A-site is regulated. SBP2 stably associates with ribosomes, and mutagenic analysis indicates that this interaction is essential for Sec incorporation. However, the ribosomal function of SBP2 has not been elucidated. To shed light on the functional relevance of the SBP2-ribosome interaction, we screened the functional centers of the 28 S rRNA in translationally competent 80 S ribosomes using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that SBP2 specifically alters the reactivity of specific residues in Helix 89 (H89) and expansion segment 31 (ES31). These results are indicative of a conformational change in response to SBP2 binding. Based on the known functions of H89 during translation, we propose that SBP2 allows Sec incorporation by either promoting Sec-tRNASec accommodation into the peptidyltransferase center and/or by stimulating the ribosome-dependent GTPase activity of eEFSec.
-
a novel protein domain induces high affinity selenocysteine insertion sequence binding and elongation factor recruitment
Journal of Biological Chemistry, 2008Co-Authors: Jesse Donovan, Kelvin Caban, Ruchira Ranaweera, Jonathan N Gonzalezflores, Paul R CopelandAbstract:Abstract Selenocysteine (Sec) is incorporated at UGA codons in mRNAs possessing a Sec insertion sequence (SECIS) Element in their 3′-untranslated region. At least three additional factors are necessary for Sec incorporation: SECIS-binding protein 2 (SBP2), Sec-tRNASec, and a Sec-specific translation elongation factor (eEFSec). The C-terminal half of SBP2 is sufficient to promote Sec incorporation in vitro, which is carried out by the concerted action of a novel Sec incorporation domain and an L7Ae RNA-binding domain. Using alanine scanning mutagenesis, we show that two distinct regions of the Sec incorporation domain are required for Sec incorporation. Physical separation of the Sec incorporation and RNA-binding domains revealed that they are able to function in trans and established a novel role of the Sec incorporation domain in promoting SECIS and eEFSec binding to the SBP2 RNA-binding domain. We propose a model in which SECIS binding induces a conformational change in SBP2 that recruits eEFSec, which in concert with the Sec incorporation domain gains access to the ribosomal A site.
-
functional analysis of the interplay between translation termination selenocysteine codon context and selenocysteine insertion sequence binding protein 2
Journal of Biological Chemistry, 2007Co-Authors: Malavika Gupta, Paul R CopelandAbstract: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.
Marla J Berry - One of the best experts on this subject based on the ideXlab platform.
-
efficient incorporation of multiple selenocysteines involves an inefficient decoding step serving as a potential translational checkpoint and ribosome bottleneck
Molecular and Cellular Biology, 2006Co-Authors: Zoia Stoytcheva, R Tujebajeva, John W Harney, Marla J BerryAbstract:Selenocysteine is incorporated into proteins via “recoding” of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3′ untranslated region, termed selenocysteine incorporation sequence (SECIS) Elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS Element, selenoprotein P genes encode multiple UGAs and two SECIS Elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a “bottleneck,” slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS Elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.
-
efficient incorporation of multiple selenocysteines involves an inefficient decoding step serving as a potential translational checkpoint and ribosome bottleneck
Molecular and Cellular Biology, 2006Co-Authors: Zoia Stoytcheva, R Tujebajeva, John W Harney, Marla J BerryAbstract:Selenocysteine is incorporated into proteins via “recoding” of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3′ untranslated region, termed selenocysteine incorporation sequence (SECIS) Elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS Element, selenoprotein P genes encode multiple UGAs and two SECIS Elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a “bottleneck,” slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS Elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.
-
decoding apparatus for eukaryotic selenocysteine insertion
EMBO Reports, 2000Co-Authors: R Tujebajeva, John W Harney, Paul R Copeland, Donna M Driscoll, Dolph L Hatfield, Xue Ming Xu, Bradley A Carlson, Marla J BerryAbstract:Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS Elements. Eukaryotic SECIS Elements are found in the 3′ untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA. Consequently, a single eukaryotic SECIS Element can serve multiple UGA codons, whereas prokaryotic SECIS Elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms. We have identified and characterized the first eukaryotic selenocysteyl-tRNA-specific elongation factor. This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells. Expression of the two functional domains of the bacterial elongation factor–SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl-tRNA–elongation factor complex, allowing a single SECIS Element to serve multiple UGA codons.
-
the caenorhabditis elegans homologue of thioredoxin reductase contains a selenocysteine insertion sequence SECIS Element that differs from mammalian SECIS Elements but directs selenocysteine incorporation
Journal of Biological Chemistry, 1999Co-Authors: Christoph Buettner, John W Harney, Marla J BerryAbstract:Thioredoxin reductases (TRR) serve critical roles in maintaining cellular redox states. Two isoforms of TRR have been identified in mammals: both contain a penultimate selenocysteine residue that is essential for catalytic activity. A search of the genome of the invertebrate, Caenorhabditis elegans, reveals a gene highly homologous to mammalian TRR, with a TGA selenocysteine codon at the corresponding position. A selenocysteyl-tRNA was identified in this organism several years ago, but no selenoproteins have been identified experimentally. Herein we report the first identification of a C. elegans selenoprotein. By (75)Se labeling of C. elegans, one major band was identified, which migrated with the predicted mobility of the C. elegans TRR homologue. Western analysis with an antibody against human TRR provides strong evidence for identification of the C. elegans selenoprotein as a member of the TRR family. The 3'-untranslated region of this gene contains a selenocysteine insertion sequence (SECIS) Element that deviates at one position from the previously invariant consensus "AUGA." Nonetheless, this Element functions to direct selenocysteine incorporation in mammalian cells, suggesting conservation of the factors recognizing SECIS Elements from worm to man.
-
knowing when not to stop selenocysteine incorporation in eukaryotes
Trends in Biochemical Sciences, 1996Co-Authors: Marla J BerryAbstract:The regulation of translation frequently involves protein-RNA interactions. An intriguing example of this is the alternative decoding of UGA, typically a stop codon, as selenocysteine. Two RNA structures, the mRNA selenocysteine insertion sequence (SECIS Element) and a unique selenocysteyl-tRNA, are required for this process. In prokaryotes, a single RNA-binding protein, a selenocysteine-specific elongation factor, interacts with both the tRNA and mRNA to confer decoding. whether eukaryotes use a similar mechanism is currently the subject of intense investigation.
Dolph L Hatfield - One of the best experts on this subject based on the ideXlab platform.
-
uga codon position dependent incorporation of selenocysteine into mammalian selenoproteins
Nucleic Acids Research, 2013Co-Authors: Anton A Turanov, Dolph L Hatfield, Alexei V Lobanov, Vadim N GladyshevAbstract:It is thought that the SelenoCysteine Insertion Sequence (SECIS) Element and UGA codon are sufficient for selenocysteine (Sec) insertion. However, we found that UGA supported Sec insertion only at its natural position or in its close proximity in mammalian thioredoxin reductase 1 (TR1). In contrast, Sec could be inserted at any tested position in mammalian TR3. Replacement of the 3 0 -UTR of TR3 with the corresponding segment of a Euplotes crassus TR restricted Sec insertion into the C-terminal region, whereas the 3 0 -UTR of TR3 conferred unrestricted Sec insertion into E. crassus TR, in which Sec insertion is normally limited to the C-terminal region. Exchanges of 3 0 -UTRs between mammalian TR1 and E. crassus TR had no effect, as both proteins restricted Sec insertion. We further found that these effects could be explained by the use of selenoprotein-specific SECIS Elements. Examination of Sec insertion into other selenoproteins was consistent with this model. The data indicate that mammals evolved the ability to limit Sec insertion into natural positions within selenoproteins, but do so in a selenoproteinspecific manner, and that this process is controlled by the SECIS Element in the 3 0 -UTR.
-
a 4 selenocysteine 2 selenocysteine insertion sequence SECIS Element methionine sulfoxide reductase from metridium senile reveals a non catalytic function of selenocysteines
Journal of Biological Chemistry, 2011Co-Authors: Alexey V Lobanov, Stefano M Marino, Alaattin Kaya, Javier Seravalli, Dolph L HatfieldAbstract:Abstract Selenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. However, how new Sec residues evolve and whether non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec insertion sequence (SECIS) Elements, one of which was a methionine-R-sulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS Elements. One of the UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas the three other UGA codons evolved recently and had no homologs with Sec or Cys in these positions. Metabolic 75Se labeling showed that all four in-frame UGA codons supported Sec insertion and that both SECIS Elements were functional and collaborated in Sec insertion at each UGA codon. Interestingly, recombinant M. senile MsrB bound iron, and further analyses suggested the possibility of binding an iron-sulfur cluster by the protein. These data show that Sec residues may appear transiently in genes containing SECIS Elements and be adapted for non-catalytic functions.
-
a highly efficient form of the selenocysteine insertion sequence Element in protozoan parasites and its use in mammalian cells
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Sergey V Novoselov, Alexey V Lobanov, Marina V Kasaikina, Dolph L HatfieldAbstract:Selenoproteins are an elite group of proteins containing a rare amino acid, selenocysteine (Sec), encoded by the codon, UGA. In eukaryotes, incorporation of Sec requires a Sec insertion sequence (SECIS) Element, a stem–loop structure located in the 3′-untranslated regions of selenoprotein mRNAs. Here we report identification of a noncanonical form of SECIS Element in Toxoplasma gondii and Neospora canine, single-celled apicomplexan parasites of humans and domestic animals. This SECIS has a GGGA sequence in the SBP2-binding site in place of AUGA previously considered invariant. Using a combination of computational and molecular techniques, we show that Toxoplasma and Neospora possess both canonical and noncanonical SECIS Elements. The GGGA-type SECIS Element supported Sec insertion in mammalian HEK 293 and NIH 3T3 cells and did so more efficiently than the natural mammalian SECIS Elements tested. In addition, mammalian type I and type II SECIS Elements mutated into the GGGA forms were functional but manifested decreased Sec insertion efficiency. We carried out computational searches for both AUGA and GGGA forms of SECIS Elements in Toxoplasma and detected five selenoprotein genes, including one coding for a previously undescribed selenoprotein, designated SelQ, and two containing the GGGA form of the SECIS Element. In contrast, the GGGA-type SECIS Elements were not detected in mammals and nematodes. As a practical outcome of the study, we developed pSelExpress1, a vector for convenient expression of selenoproteins in mammalian cells. It contains an SBP2 gene and the most efficient tested SECIS Element: an AUGA mutant of the GGGA-type Toxoplasma SelT structure.
-
a highly efficient form of the selenocysteine insertion sequence Element in protozoan parasites and its use in mammalian cells
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Sergey V Novoselov, Alexey V Lobanov, Marina V Kasaikina, Dolph L Hatfield, Vadim N GladyshevAbstract:Selenoproteins are an elite group of proteins containing a rare amino acid, selenocysteine (Sec), encoded by the codon, UGA. In eukaryotes, incorporation of Sec requires a Sec insertion sequence (SECIS) Element, a stem–loop structure located in the 3′-untranslated regions of selenoprotein mRNAs. Here we report identification of a noncanonical form of SECIS Element in Toxoplasma gondii and Neospora canine, single-celled apicomplexan parasites of humans and domestic animals. This SECIS has a GGGA sequence in the SBP2-binding site in place of AUGA previously considered invariant. Using a combination of computational and molecular techniques, we show that Toxoplasma and Neospora possess both canonical and noncanonical SECIS Elements. The GGGA-type SECIS Element supported Sec insertion in mammalian HEK 293 and NIH 3T3 cells and did so more efficiently than the natural mammalian SECIS Elements tested. In addition, mammalian type I and type II SECIS Elements mutated into the GGGA forms were functional but manifested decreased Sec insertion efficiency. We carried out computational searches for both AUGA and GGGA forms of SECIS Elements in Toxoplasma and detected five selenoprotein genes, including one coding for a previously undescribed selenoprotein, designated SelQ, and two containing the GGGA form of the SECIS Element. In contrast, the GGGA-type SECIS Elements were not detected in mammals and nematodes. As a practical outcome of the study, we developed pSelExpress1, a vector for convenient expression of selenoproteins in mammalian cells. It contains an SBP2 gene and the most efficient tested SECIS Element: an AUGA mutant of the GGGA-type Toxoplasma SelT structure.
-
distribution and functional consequences of nucleotide polymorphisms in the 3 untranslated region of the human sep15 gene
Cancer Research, 2001Co-Authors: Ya Jun Hu, Dolph L Hatfield, Konstantin V Korotkov, Rajeshwari R Mehta, Charles N Rotimi, Amy Luke, Elaine T Prewitt, Richard Cooper, Wendy Stock, Everett E VokesAbstract:Selenium has been shown to prevent cancer in a variety of animal model systems. Both epidemiological studies and supplementation trials have supported its efficacy in humans. However, the mechanism by which selenium suppresses tumor development remains unknown. Selenium is present in known human selenoproteins as the amino acid selenocysteine (Sec). Sec is inserted cotranslationally in response to UGA codons within selenoprotein mRNAs in a process requiring a sequence within the 3′-untranslated region (UTR), referred to as a Sec insertion sequence (SECIS) Element. Recently, a human M r 15,000 selenoprotein ( Sep15 ) was identified that contains an in-frame UGA codon and a SECIS Element in the 3′-UTR. Examination of the available cDNA sequences for this protein revealed two polymorphisms located at position 811 (C/T) and at position 1125 (G/A) located within the 3′-UTR. Here, we demonstrate significant differences in Sep15 allele frequencies by ethnicity and that the identity of the nucleotides at the polymorphic sites influences SECIS function in a selenium-dependent manner. This, together with genetic data indicating loss of heterozygosity at the Sep15 locus in certain human tumor types, suggests that Sep15 may be involved in cancer development, risk, or both.
John W Harney - One of the best experts on this subject based on the ideXlab platform.
-
efficient incorporation of multiple selenocysteines involves an inefficient decoding step serving as a potential translational checkpoint and ribosome bottleneck
Molecular and Cellular Biology, 2006Co-Authors: Zoia Stoytcheva, R Tujebajeva, John W Harney, Marla J BerryAbstract:Selenocysteine is incorporated into proteins via “recoding” of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3′ untranslated region, termed selenocysteine incorporation sequence (SECIS) Elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS Element, selenoprotein P genes encode multiple UGAs and two SECIS Elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a “bottleneck,” slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS Elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.
-
efficient incorporation of multiple selenocysteines involves an inefficient decoding step serving as a potential translational checkpoint and ribosome bottleneck
Molecular and Cellular Biology, 2006Co-Authors: Zoia Stoytcheva, R Tujebajeva, John W Harney, Marla J BerryAbstract:Selenocysteine is incorporated into proteins via “recoding” of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3′ untranslated region, termed selenocysteine incorporation sequence (SECIS) Elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS Element, selenoprotein P genes encode multiple UGAs and two SECIS Elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a “bottleneck,” slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS Elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.
-
decoding apparatus for eukaryotic selenocysteine insertion
EMBO Reports, 2000Co-Authors: R Tujebajeva, John W Harney, Paul R Copeland, Donna M Driscoll, Dolph L Hatfield, Xue Ming Xu, Bradley A Carlson, Marla J BerryAbstract:Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS Elements. Eukaryotic SECIS Elements are found in the 3′ untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA. Consequently, a single eukaryotic SECIS Element can serve multiple UGA codons, whereas prokaryotic SECIS Elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms. We have identified and characterized the first eukaryotic selenocysteyl-tRNA-specific elongation factor. This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells. Expression of the two functional domains of the bacterial elongation factor–SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl-tRNA–elongation factor complex, allowing a single SECIS Element to serve multiple UGA codons.
-
the caenorhabditis elegans homologue of thioredoxin reductase contains a selenocysteine insertion sequence SECIS Element that differs from mammalian SECIS Elements but directs selenocysteine incorporation
Journal of Biological Chemistry, 1999Co-Authors: Christoph Buettner, John W Harney, Marla J BerryAbstract:Thioredoxin reductases (TRR) serve critical roles in maintaining cellular redox states. Two isoforms of TRR have been identified in mammals: both contain a penultimate selenocysteine residue that is essential for catalytic activity. A search of the genome of the invertebrate, Caenorhabditis elegans, reveals a gene highly homologous to mammalian TRR, with a TGA selenocysteine codon at the corresponding position. A selenocysteyl-tRNA was identified in this organism several years ago, but no selenoproteins have been identified experimentally. Herein we report the first identification of a C. elegans selenoprotein. By (75)Se labeling of C. elegans, one major band was identified, which migrated with the predicted mobility of the C. elegans TRR homologue. Western analysis with an antibody against human TRR provides strong evidence for identification of the C. elegans selenoprotein as a member of the TRR family. The 3'-untranslated region of this gene contains a selenocysteine insertion sequence (SECIS) Element that deviates at one position from the previously invariant consensus "AUGA." Nonetheless, this Element functions to direct selenocysteine incorporation in mammalian cells, suggesting conservation of the factors recognizing SECIS Elements from worm to man.
-
the 3 untranslated region of human type 2 iodothyronine deiodinase mrna contains a functional selenocysteine insertion sequence Element
Journal of Biological Chemistry, 1998Co-Authors: Christoph Buettner, John W Harney, Reed P LarsenAbstract:Type 2 deiodinase (D2) catalyzes the 5′-deiodination of thyroxine to form 3,5,3′-triiodothyronine. Two mammalian D2 cDNAs have been identified containing 2 kilobases (kb) of the 7-kb mRNA including the complete coding sequence. Both contain in-frame TGA codons, which should serve as selenocysteine codons. However, the selenocysteine insertion sequence (SECIS) Elements required for the decoding of UGA as a selenocysteine in the 3′-untranslated region (UTR) of the mRNA are not present. We have identified two overlapping expressed sequence tag clones, which contain the missing 4.4-kb 3′-UTR of the human D2 (hD2) cDNA. Computer analysis predicts a stem loop structure 280 base pairs 5′ to the polyadenylation site, which has potent SECIS activity. A fragment containing these sequences hybridizes to D2 mRNA in human thyroid. A G to A mutation in the essential AUGA motif of this Element abolished its function. Transfection of the hD2 coding region plus the 3′-UTR results in the expression of D2, and its in vitrotranscribed mRNA programs D2 activity in Xenopusoocytes. This is the first identification of a SECIS Element in a mammalian D2 cDNA and establishes that hD2 is a bona fide selenoprotein.