Mutations disrupting selenocysteine formation cause progressive cerebello-cerebral atrophy

The essential micronutrient selenium is found in proteins as selenocysteine (Sec), the only genetically encoded amino acid whose biosynthesis occurs on its cognate tRNA in humans. In the final step of selenocysteine formation, the essential enzyme SepSecS catalyzes the conversion of Sep-tRNA to Sec-tRNA. We demonstrate that SepSecS mutations cause autosomal-recessive progressive cerebellocerebral atrophy (PCCA) in Jews of Iraqi and Moroccan ancestry. Both founder mutations, common in these two populations, disrupt the sole route to the biosynthesis of the 21st amino acid, Sec, and thus to the generation of selenoproteins in humans.     

New Developments in Selenium Biochemistry: Selenocysteine Biosynthesis in Eukaryotes and Archaea

We used comparative genomics and experimental analyses to show that (1) eukaryotes and archaea, which possess the selenocysteine (Sec) protein insertion machinery contain an enzyme, O-phosphoseryl-transfer RNA (tRNA)^[Ser]Sec kinase (designated PSTK), which phosphorylates seryl-tRNA^[Ser]Sec to form O-phosphoseryl-tRNA^[Ser]Sec and (2) the Sec synthase (SecS) in mammals is a pyridoxal phosphate-containing protein previously described as the soluble liver antigen (SLA). SecS uses the product of PSTK, O-phosphoseryl-tRNA^[Ser]Sec, and selenophosphate as substrates to generate selenocysteyl-tRNA^[Ser]Sec. Sec could be synthesized on tRNA^[Ser]Sec from selenide, adenosine triphosphate (ATP), and serine using tRNA^[Ser]Sec, seryl-tRNA synthetase, PSTK, selenophosphate synthetase, and SecS. The enzyme that synthesizes monoselenophosphate is a previously identified selenoprotein, selenophosphate synthetase 2 (SPS2), whereas the previously identified mammalian selenophosphate synthetase 1 did not serve this function. Monoselenophosphate also served directly in the reaction replacing ATP, selenide, and SPS2, demonstrating that this compound was the active selenium donor. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that contain selenoproteins. X.-M. Xu and B. A. Carlson contributed equally to the studies described herein.     

Divergence of selenocysteine tRNA recognition by archaeal and eukaryotic O-phosphoseryl-tRNASec kinase

Selenocysteine (Sec) biosynthesis in archaea and eukaryotes requires three steps: serylation of tRNA^Sec by seryl-tRNA synthetase (SerRS), phosphorylation of Ser-tRNA^Sec by O-phosphoseryl-tRNA^Sec kinase (PSTK), and conversion of O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) by Sep-tRNA:Sec-tRNA synthase (SepSecS) to Sec-tRNA^Sec. Although SerRS recognizes both tRNA^Sec and tRNA^Ser species, PSTK must discriminate Ser-tRNA^Sec from Ser-tRNA^Ser. Based on a comparison of the sequences and secondary structures of archaeal tRNA^Sec and tRNA^Ser, we introduced mutations into Methanococcus maripaludis tRNA^Sec to investigate how Methanocaldococcus jannaschii PSTK distinguishes tRNA^Sec from tRNA^Ser. Unlike eukaryotic PSTK, the archaeal enzyme was found to recognize the acceptor stem rather than the length and secondary structure of the D-stem. While the D-arm and T-loop provide minor identity elements, the acceptor stem base pairs G2-C71 and C3-G70 in tRNA^Sec were crucial for discrimination from tRNA^Ser. Furthermore, the A5-U68 base pair in tRNA^Ser has some antideterminant properties for PSTK. Transplantation of these identity elements into the tRNA^Ser_UGA scaffold resulted in phosphorylation of the chimeric Ser-tRNA. The chimera was able to stimulate the ATPase activity of PSTK albeit at a lower level than tRNA^Sec, whereas tRNA^Ser did not. Additionally, the seryl moiety of Ser-tRNA^Sec is not required for enzyme recognition, as PSTK efficiently phosphorylated Thr-tRNA^Sec.     

Evolution of selenium utilization traits

Background  

The essential trace element selenium is used in a wide variety of biological processes. Selenocysteine (Sec), the 21st amino acid, is co-translationally incorporated into a restricted set of proteins. It is encoded by an UGA codon with the help of tRNA^Sec (SelC), Sec-specific elongation factor (SelB) and a cis-acting mRNA structure (SECIS element). In addition, Sec synthase (SelA) and selenophosphate synthetase (SelD) are involved in the biosynthesis of Sec on the tRNA^Sec. Selenium is also found in the form of 2-selenouridine, a modified base present in the wobble position of certain tRNAs, whose synthesis is catalyzed by YbbB using selenophosphate as a precursor.     

Selenophosphate synthetase 2 is essential for selenoprotein biosynthesis

Selenophosphate synthetase (SelD) generates the selenium donor for selenocysteine biosynthesis in eubacteria. One homologue of SelD in eukaryotes is SPS1 (selenophosphate synthetase 1) and a second one, SPS2, was identified as a selenoprotein in mammals. Earlier in vitro studies showed SPS2, but not SPS1, synthesized selenophosphate from selenide, whereas SPS1 may utilize a different substrate. The roles of these enzymes in selenoprotein synthesis in vivo remain unknown. To address their function in vivo, we knocked down SPS2 in NIH3T3 cells using small interfering RNA and found that selenoprotein biosynthesis was severely impaired, whereas knockdown of SPS1 had no effect. Transfection of SPS2 into SPS2 knockdown cells restored selenoprotein biosynthesis, but SPS1 did not, indicating that SPS1 cannot complement SPS2 function. These in vivo studies indicate that SPS2 is essential for generating the selenium donor for selenocysteine biosynthesis in mammals, whereas SPS1 probably has a more specialized, non-essential role in selenoprotein metabolism.     

Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation

Selenocysteine (Sec)-decoding archaea and eukaryotes employ a unique route of Sec-tRNA^Sec synthesis in which O-phosphoseryl-tRNA^Sec kinase (PSTK) phosphorylates Ser-tRNA^Sec to produce the O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) substrate that Sep-tRNA:Sec-tRNA synthase (SepSecS) converts to Sec-tRNA^Sec. This study presents a biochemical characterization of Methanocaldococcus jannaschii PSTK, including kinetics of Sep-tRNA^Sec formation (K_m for Ser-tRNA^Sec of 40 nM and ATP of 2.6 mM). PSTK binds both Ser-tRNA^Sec and tRNA^Sec with high affinity (K_d values of 53 nM and 39 nM, respectively). The ATPase activity of PSTK may be activated via an induced fit mechanism in which binding of tRNA^Sec specifically stimulates hydrolysis. Albeit with lower activity than ATP, PSTK utilizes GTP, CTP, UTP and dATP as phosphate-donors. Homology with related kinases allowed prediction of the ATPase active site, comprised of phosphate-binding loop (P-loop), Walker B and RxxxR motifs. Gly14, Lys17, Ser18, Asp41, Arg116 and Arg120 mutations resulted in enzymes with decreased activity highlighting the importance of these conserved motifs in PSTK catalysis both in vivo and in vitro. Phylogenetic analysis of PSTK in the context of its ‘DxTN’ kinase family shows that PSTK co-evolved precisely with SepSecS and indicates the presence of a previously unidentified PSTK in Plasmodium species.     

In vivo requirement of selenophosphate for selenoprotein synthesis in archaea

Biosynthesis of selenocysteine, the 21st proteinogenic amino acid, occurs bound to a dedicated tRNA in all three domains of life, Bacteria, Eukarya and Archaea, but differences exist between the mechanism employed by bacteria and eukaryotes/archaea. The role of selenophosphate and the enzyme providing it, selenophosphate synthetase, in archaeal selenoprotein synthesis was addressed by mutational analysis. Surprisingly, MMP0904, encoding a homologue of eukaryal selenophosphate synthetase in Methanococcus maripaludis S2, could not be deleted unless selD , encoding selenophosphate synthetase of Escherichia coli , was present in trans , demonstrating that the factor is essential for the organism. In contrast, the homologous gene of M. maripaludis JJ could be readily deleted, obviating the strain's ability to synthesize selenoproteins. Complementing with selD restored selenoprotein synthesis, demonstrating that the deleted gene encodes selenophosphate synthetase and that selenophosphate is the in vivo selenium donor for selenoprotein synthesis of this organism. We also showed that this enzyme is a selenoprotein itself and that M. maripaludis contains another, HesB-like selenoprotein previously only predicted from genome analyses. The data highlight the use of genetic methods in archaea for a causal analysis of their physiology and, by comparing two closely related strains of the same species, illustrate the evolution of the selenium-utilizing trait.     

From one amino acid to another: tRNA-dependent amino acid biosynthesis

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms.     

A selDABC cluster for selenocysteine incorporation in Eubacterium acidaminophilum

The four genes required for selenocysteine incorporation were isolated from the gram-positive, amino acid-fermenting anaerobe Eubacterium acidaminophilum, which expresses various selenoproteins of different functions. The sel genes were located in an unique organization on a continuous fragment of genomic DNA in the order selD1 (selenophosphate synthetase 1), selA (selenocysteine synthase), selB (selenocysteine-specific elongation factor), and selC (selenocysteine-specific tRNA). A second gene copy, encoding selenophosphate synthetase 2 (selD2), was present on a separate fragment of genomic DNA. SelD1 and SelD2 were only 62.9% identical, but the two encoding genes, selD1 and selD2, contained an in-frame UGA codon encoding selenocysteine, which corresponds to Cys-17 of Escherichia coli SelD. The function of selA, selB, and selC from E. acidaminophilum was investigated by complementation of the respective E. coli deletion mutant strains and determined as the benzyl viologen-dependent formate dehydrogenase activity in these strains after anaerobic growth in the presence of formate. selA and selC from E. acidaminophilum were functional and complemented the respective mutant strains to 83% (selA) and 57% (selC) compared to a wild-type strain harboring the same plasmid. Complementation of the E. coli selB mutant was only observed when both selB and selC from E. acidaminophilum were present. Under these conditions, the specific activity of formate dehydrogenase was 55% of that of the wild type. Transformation of this selB mutant with selB alone was not sufficient to restore formate dehydrogenase activity.     

Biosynthesis of selenocysteine on its tRNA in eukaryotes

Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA^[Ser]Sec as substrates to generate selenocysteyl-tRNA^[Ser]Sec. Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA^[Ser]Sec, seryl-tRNA synthetase, O-phosphoseryl-tRNA^[Ser]Sec kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA^[Ser]Sec kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins.     

Selenoproteinless animals: selenophosphate synthetase SPS1 functions in a pathway unrelated to selenocysteine biosynthesis

Proteins containing the 21st amino acid, selenocysteine (Sec), have been described in all three domains of life, but the composition of selenoproteomes in organisms varies significantly. Here, we report that aquatic arthropods possess many selenoproteins also detected in other animals and unicellular eukaryotes, and that most of these proteins were either lost or replaced with cysteine-containing homologs in insects. As a result of this selective selenoproteome reduction, fruit flies and mosquitoes have three known selenoproteins, and the honeybee, Apis mellifera, a single detected candidate selenoprotein. Moreover, we identified the red flour beetle, Tribolium castaneum, and the silkworm, Bombyx mori, as the first animals that lack any Sec-containing proteins. These insects also lost the Sec biosynthesis and insertion machinery, but selenophosphate synthetase 1 (SPS1), an enzyme previously implicated in Sec biosynthesis, is present in all insects, including T. castaneum and B. mori. These data indicate that SPS1 functions in a pathway unrelated to selenoprotein synthesis. Since SPS1 evolved from a protein that utilizes selenium for Sec biosynthesis, an attractive possibility is that SPS1 may define a new pathway of selenium utilization in animals.     

Evolution of selenium utilization traits

Background

The essential trace element selenium is used in a wide variety of biological processes. Selenocysteine (Sec), the 21st amino acid, is co-translationally incorporated into a restricted set of proteins. It is encoded by an UGA codon with the help of tRNA^Sec (SelC), Sec-specific elongation factor (SelB) and a cis-acting mRNA structure (SECIS element). In addition, Sec synthase (SelA) and selenophosphate synthetase (SelD) are involved in the biosynthesis of Sec on the tRNA^Sec. Selenium is also found in the form of 2-selenouridine, a modified base present in the wobble position of certain tRNAs, whose synthesis is catalyzed by YbbB using selenophosphate as a precursor.

Results

We analyzed completely sequenced genomes for occurrence of the selA, B, C, D and ybbB genes. We found that selB and selC are gene signatures for the Sec-decoding trait. However, selD is also present in organisms that do not utilize Sec, and shows association with either selA, B, C and/or ybbB. Thus, selD defines the overall selenium utilization. A global species map of Sec-decoding and 2-selenouridine synthesis traits is provided based on the presence/absence pattern of selenium-utilization genes. The phylogenies of these genes were inferred and compared to organismal phylogenies, which identified horizontal gene transfer (HGT) events involving both traits.

Conclusion

These results provide evidence for the ancient origin of these traits, their independent maintenance, and a highly dynamic evolutionary process that can be explained as the result of speciation, differential gene loss and HGT. The latter demonstrated that the loss of these traits is not irreversible as previously thought.     

The class 2 selenophosphate synthetase gene of Drosophila contains a functional mammalian-type SECIS

Synthesis of monoselenophosphate, the selenium donor required for the synthesis of selenocysteine (Sec) is catalyzed by the enzyme selenophosphate synthetase (SPS), first described in Escherichia coli. SPS homologs were identified in archaea, mammals and Drosophila. In the latter, however, an amino acid replacement is present within the catalytic domain and lacks selenide-dependent SPS activity. We describe the identification of a novel Drosophila homolog, Dsps2. The open reading frame of Dsps2 mRNA is interrupted by an UGA stop codon. The 3'UTR contains a mammalian-like Sec insertion sequence which causes translational readthrough in both transfected Drosophila cells and transgenic embryos. Thus, like vertebrates, Drosophila contains two SPS enzymes one with and one without Sec in its catalytic domain. Our data indicate further that the selenoprotein biosynthesis machinery is conserved between mammals and fly, promoting the use of Drosophila as a genetic tool to identify components and mechanistic features of the synthesis pathway.     

Amino acid modifications on tRNA

The accurate formation of cognate aminoacyl-transfer RNAs (aa-tRNAs) is essential for the fidelity of translation. Most amino acids are esterified onto their cognate tRNA isoacceptors directly by aa-tRNA synthetases. However, in the case of four amino acids (Gln, Asn, Cys and Sec), aminoacyl-tRNAs are made through indirect pathways in many organisms across all three domains of life. The process begins with the charging of noncognate amino acids to tRNAs by a specialized synthetase in the case of Cys-tRNA(Cys) formation or by synthetases with relaxed specificity, such as the non-discriminating glutamyl-tRNA, non-discriminating aspartyl-tRNA and seryl-tRNA synthetases. The resulting misacylated tRNAs are then converted to cognate pairs through transformation of the amino acids on the tRNA, which is catalyzed by a group of tRNA-dependent modifying enzymes, such as tRNA-dependent amidotransferases, Sep-tRNA:Cys-tRNA synthase, O-phosphoseryl-tRNA kinase and Sep-tRNA:Sec-tRNA synthase. The majority of these indirect pathways are widely spread in all domains of life and thought to be part of the evolutionary process.     

A tRNA-dependent cysteine biosynthesis enzyme recognizes the selenocysteine-specific tRNA in Escherichia coli

The essential methanogen enzyme Sep-tRNA:Cys-tRNA synthase (SepCysS) converts O-phosphoseryl-tRNA^Cys (Sep-tRNA^Cys) into Cys-tRNA^Cys in the presence of a sulfur donor. Likewise, Sep-tRNA:Sec-tRNA synthase converts O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) to selenocysteinyl-tRNA^Sec (Sec-tRNA^Sec) using a selenium donor. While the Sep moiety of the aminoacyl-tRNA substrates is the same in both reactions, tRNA^Cys and tRNA^Sec differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that Sep-tRNA^Sec is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep-tRNA^Sec to Cys-tRNA^Sec, and that Sep is crucial for SepCysS recognition.     

Selenoprotein synthesis in archaea

The availability of the genome sequences from several archaea has facilitated the identification of the encoded selenoproteins and also of most of the components of the machinery for selenocysteine biosynthesis and insertion. Until now, selenoproteins have been identified solely in species of the genera Methanococcus (M.) and Methanopyrus. Apart from selenophosphate synthetase, they include only enzymes with a function in energy metabolism. Like in bacteria and eukarya, selenocysteine insertion is directed by a UGA codon in the mRNA and involves the action of a specific tRNA and of selenophosphate as the selenium donor. Major differences to the bacterial system, however, are that no homolog for the bacterial selenocysteine synthase was found and, especially, that the SECIS element of the mRNA is positioned in the 3' nontranslated region. The characterisation of a homolog for the bacterial SelB protein showed that it does not bind to the SECIS element necessitating the activity of at least a second protein. The use of the genetic system of M. maripaludis allowed the heterologous expression of a selenoprotein gene from M. jannaschii and will facilitate the elucidation of the mechanism of the selenocysteine insertion process in the future.     

Utilization of selenocysteine as a source of selenium for selenophosphate biosynthesis

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate from selenide and ATP. Characterization of selenophosphate synthetase revealed the determined K(m) value for selenide is far above the optimal concentration needed for growth and approached levels which are toxic. Selenocysteine lyase enzymes, which decompose selenocysteine to elemental selenium (Se(0)) and alanine, were considered as candidates for the control of free selenium levels in vivo. The ability of a lyase protein to generate Se(0) in the proximity of SPS maybe an attractive solution to selenium toxicity as well as the high K(m) value for selenide. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, were characterized. All three proteins exhibit lyase activity on L-cysteine and L-selenocysteine and produce sulfane sulfur, S(0), or Se(0) respectively. Each lyase can effectively mobilize Se(0) from L-selenocysteine for selenophosphate biosynthesis.