Cerebellar Hypoplasia in Mice Lacking Selenoprotein Biosynthesis in Neurons

Selenium exerts many, if not most, of its physiological functions as a selenocysteine moiety in proteins. Selenoproteins are involved in many biochemical processes including regulation of cellular redox state, calcium homeostasis, protein biosynthesis, and degradation. A neurodevelopmental syndrome called progressive cerebello-cortical atrophy (PCCA) is caused by mutations in the selenocysteine synthase gene, SEPSECS, demonstrating that selenoproteins are essential for human brain development. While we have shown that selenoproteins are required for correct hippocampal and cortical interneuron development, little is known about the functions of selenoproteins in the cerebellum. Therefore, we have abrogated neuronal selenoprotein biosynthesis by conditional deletion of the gene encoding selenocysteyl tRNA^[Ser]Sec (gene symbol Trsp). Enzymatic activity of cellular glutathione peroxidase and cytosolic thioredoxin reductase is reduced in cerebellar extracts from Trsp-mutant mice. These mice grow slowly and fail to gain postural control or to coordinate their movements. Histological analysis reveals marked cerebellar hypoplasia, associated with Purkinje cell death and decreased granule cell proliferation. Purkinje cell death occurs along parasagittal stripes as observed in other models of Purkinje cell loss. Neuron-specific inactivation of glutathione peroxidase 4 (Gpx4) used the same Cre driver phenocopies tRNA^[Ser]Sec mutants in several aspects: cerebellar hypoplasia, stripe-like Purkinje cell loss, and reduced granule cell proliferation. Parvalbumin-expressing GABAergic interneurons (stellate and/or basket cells) are virtually absent in tRNA^[Ser]Sec-mutant mice, while some remained in Gpx4-mutant mice. Our data show that selenoproteins are specifically required in postmitotic neurons of the developing cerebellum, thus providing a rational explanation for cerebellar hypoplasia as occurring in PCCA patients.     

Loss of housekeeping selenoprotein expression in mouse liver modulates lipoprotein metabolism

Selenium is incorporated into proteins as selenocysteine (Sec), which is dependent on its specific tRNA, designated tRNA^[Ser]Sec. Targeted removal of the tRNA^[Ser]Sec gene (Trsp) in mouse hepatocytes previously demonstrated the importance of selenoproteins in liver function. Herein, analysis of plasma proteins in this Trsp knockout mouse revealed increases in apolipoprotein E (ApoE) that was accompanied by elevated plasma cholesterol levels. The expression of genes involved in cholesterol biosynthesis, metabolism and transport were also altered in knockout mice. Additionally, in two transgenic Trsp mutant mouse lines (wherein only housekeeping selenoprotein synthesis was restored), the expression of ApoE, as well as genes involved in cholesterol biosynthesis, metabolism and transport were similar to those observed in wild type mice. These data correlate with reports that selenium deficiency results in increased levels of ApoE, indicating for the first time that housekeeping selenoproteins have a role in regulating lipoprotein biosynthesis and metabolism.     

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.     

Human Cells Have a Limited Set of tRNA Anticodon Loop Substrates of the tRNA Isopentenyltransferase TRIT1 Tumor Suppressor

Human TRIT1 is a tRNA isopentenyltransferase (IPTase) homologue of Escherichia coli MiaA, Saccharomyces cerevisiae Mod5, Schizosaccharomyces pombe Tit1, and Caenorhabditis elegans GRO-1 that adds isopentenyl groups to adenosine 37 (i6A37) of substrate tRNAs. Prior studies indicate that i6A37 increases translation fidelity and efficiency in codon-specific ways. TRIT1 is a tumor suppressor whose mutant alleles are associated with cancer progression. We report the systematic identification of i6A37-containing tRNAs in a higher eukaryote, performed using small interfering RNA knockdown and other methods to examine TRIT1 activity in HeLa cells. Although several potential substrates contained the IPTase recognition sequence A36A37A38 in the anticodon loop, only tRNA^SerAGA, tRNA^SerCGA, tRNA^SerUGA, and selenocysteine tRNA with UCA (tRNA^[Ser]SecUCA) contained i6A37. This subset is a significantly more restricted than that for two distant yeasts (S. cerevisiae and S. pombe), the only other organisms comprehensively examined. Unlike the fully i6A37-modified tRNAs for Ser, tRNA^[Ser]SecUCA is partially (∼40%) modified. Exogenous selenium and other treatments that decreased the i6A37 content of tRNA^[Ser]SecUCA led to increased levels of the tRNA^[Ser]SecUCA. Of the human mitochondrion (mt)-encoded tRNAs with A36A37A38, only mt tRNAs tRNA^SerUGA and tRNA^TrpUCA contained detectable i6A37. Moreover, while tRNA^Ser levels were unaffected by TRIT1 knockdown, the tRNA^[Ser]SecUCA level was increased and the mt tRNA^SerUGA level was decreased, suggesting that TRIT1 may control the levels of some tRNAs as well as their specific activity.     

大肠杆菌tRNASec关键核苷酸位点

在原核生物中,硒蛋白合成需要tRNA~(Sec) (SelC)与硒代半胱氨酸合成(Sec synthase, SelA)、硒代半胱氨酸特异性延伸因子(Sec-specificelongationfactor,SelB)之间相互作用。【目的】基于大肠杆菌掺硒机器,寻找tRNA~(Sec)骨架上关键核苷酸位点,为解决硒蛋白目前面临的掺硒效率较低、产量低的问题提供新思路。【方法】以大鼠细胞质型硫氧还蛋白还原酶(thioredoxinreductase1,TrxR1)为掺硒模式蛋白为定点突变tRNA~(Sec),转化至BL21 (DE3) gor-获得阳性重组菌株(携带pET-TRSter/pSUABC’),用于表达大鼠硒蛋白TrxR1,然后使用2￠,5￠ADP-Sepharose亲和层析和凝胶过滤两步法分离纯化TrxR1,最后利用经典硒依赖型DTNB还原反应测定TrxR1的酶活,分析关键核苷酸位点,评价掺硒效率。【结果】在存在SECIS元件的前提下,当SelA、SelB、tRNA~(Sec)共表达时,与野生型相比,携带突变型tRNA~(Sec)所共表达的TrxR1酶活力呈现不同程度的降低,其中E.colitRNA~(Sec)的G18、G19这两个位点的所有的TrxR1酶活远低于野生型(10%);然而,a26和b7的酶活相对较高。【结论】E. coli tRNA~(Sec)骨架上G18和G19位点对于维持tRNA稳定性和灵活性发挥了关键作用,位点突变引起tRNA结构变化会影响tRNA~(Sec)与掺硒元件的互作,因此有望通过改造tRNA核苷酸位点来提高硒蛋白的掺硒效率。     

Evidence for direct roles of two additional factors, SECp43 and soluble liver antigen, in the selenoprotein synthesis machinery

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2'-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA.     

Inhibition of Cellular Methyltransferases Promotes Endothelial Cell Activation by Suppressing Glutathione Peroxidase 1 Protein Expression

S-Adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNA^Sec to the Um34 form. The formation of methylated tRNA^Sec facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNA^Sec and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNA^Sec hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype.     

Supramolecular complexes mediate selenocysteine incorporation in vivo

Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA([Ser]Sec) and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA([Ser]Sec), termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization. We further show that SECp43 interacts with the selenocysteyl-tRNA([Ser]Sec)-EFsec complex in vitro, and SECp43 coexpression promotes interaction between EFsec and SBP2 in vivo. Additionally, SECp43 increases selenocysteine incorporation and selenoprotein mRNA levels, the latter presumably due to circumvention of nonsense-mediated decay. Thus, SECp43 emerges as a key player in orchestrating the interactions and localization of the other factors involved in selenoprotein biosynthesis. Finally, our studies delineating the multiple, coordinated protein-nucleic acid interactions between SECp43 and the previously described selenoprotein cotranslational factors resulted in a model of selenocysteine biosynthesis and incorporation dependent upon both cytoplasmic and nuclear supramolecular complexes.     

Osteo-Chondroprogenitor–Specific Deletion of the Selenocysteine tRNA Gene,Trsp, Leads to Chondronecrosis and Abnormal Skeletal Development: A Putative Model for Kashin-Beck Disease

Kashin-Beck disease, a syndrome characterized by short stature, skeletal deformities, and arthropathy of multiple joints, is highly prevalent in specific regions of Asia. The disease has been postulated to result from a combination of different environmental factors, including contamination of barley by mold mycotoxins, iodine deficiency, presence of humic substances in drinking water, and, importantly, deficiency of selenium. This multifunctional trace element, in the form of selenocysteine, is essential for normal selenoprotein function, including attenuation of excessive oxidative stress, and for the control of redox-sensitive molecules involved in cell growth and differentiation. To investigate the effects of skeletal selenoprotein deficiency, a Cre recombinase transgenic mouse line was used to trigger Trsp gene deletions in osteo-chondroprogenitors. Trsp encodes selenocysteine tRNA^[Ser]Sec, required for the incorporation of selenocysteine residues into selenoproteins. The mutant mice exhibited growth retardation, epiphyseal growth plate abnormalities, and delayed skeletal ossification, as well as marked chondronecrosis of articular, auricular, and tracheal cartilages. Phenotypically, the mice thus replicated a number of the pathological features of Kashin-Beck disease, supporting the notion that selenium deficiency is important to the development of this syndrome.     

Structural Asymmetry of the Terminal Catalytic Complex in Selenocysteine Synthesis

Selenocysteine (Sec), the 21^st amino acid, is synthesized from a serine precursor in a series of reactions that require selenocysteine tRNA (tRNA^Sec). In archaea and eukaryotes, O-phosphoseryl-tRNA^Sec:selenocysteinyl-tRNA^Sec synthase (SepSecS) catalyzes the terminal synthetic reaction during which the phosphoseryl intermediate is converted into the selenocysteinyl moiety while being attached to tRNA^Sec. We have previously shown that only the SepSecS tetramer is capable of binding to and recognizing the distinct fold of tRNA^Sec. Because only two of the four tRNA-binding sites were occupied in the crystal form, a question was raised regarding whether the observed arrangement and architecture faithfully recapitulated the physiologically relevant ribonucleoprotein complex important for selenoprotein formation. Herein, we determined the stoichiometry of the human terminal synthetic complex of selenocysteine by using small angle x-ray scattering, multi-angle light scattering, and analytical ultracentrifugation. In addition, we provided the first estimate of the ratio between SepSecS and tRNA^Sec in vivo. We show that SepSecS preferentially binds one or two tRNA^Sec molecules at a time and that the enzyme is present in large molar excess over the substrate tRNA in vivo. Moreover, we show that in a complex between SepSecS and two tRNAs, one enzyme homodimer plays a role of the noncatalytic unit that positions CCA ends of two tRNA^Sec molecules into the active site grooves of the other, catalytic, homodimer. Finally, our results demonstrate that the previously determined crystal structure represents the physiologically and catalytically relevant complex and suggest that allosteric regulation of SepSecS might play an important role in regulation of selenocysteine and selenoprotein synthesis.     

Role of stoichiometry between mRNA, translation factor SeIB and selenocysteyl-tRNA in selenoprotein synthesis

The specialized translation factor SeIB forms a quaternary complex in vitro with selenocysteyl-tRNA^Sec, the selenoprotein mRNA and guanine nucleotides. To gain information on whether this complex is required for selenocysteine insertion in vivo we have studied the effect of unbalanced ratios of the individual components of the complex on UGA readthrough. It was found that overproduction of SeIB in an otherwise wild-type genetic background reduced UGA read-through to less than 1 %. Concomitant overexpression of seIC (the gene for selenocysteine-specific tRNA^Sec) completely reversed the inhibition. Truncation of SeIB from the C-terminal end abolished function as a translation factor but the truncated molecules, when overproduced, were still able to suppress UGA read-through. The inhibition was also reversed by overproduction of tRNA^Sec. The most plausible explanation is that overproduction of SeIB impairs the statistics of formation of the quaternary complex and that the C-terminally truncated molecules are still able to bind selenocysteyl-tRNA^Sec and remove it from the pool. The mRNA-binding capacity, therefore, is physically separated from the selenocysteyl-tRNA-binding domain.     

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.     

The Specific Elongation Factor to Selenocysteine Incorporation in Escherichia coli: Unique tRNASec Recognition and its Interactions

Several molecular mechanisms are involved in the genetic code interpretation during translation, as codon degeneration for the incorporation of rare amino acids. One mechanism that stands out is selenocysteine (Sec), which requires a specific biosynthesis and incorporation pathway. In Bacteria, the Sec biosynthesis pathway has unique features compared with the eukaryote pathway as Ser to Sec conversion mechanism is accomplished by a homodecameric enzyme (selenocysteine synthase, SelA) followed by the action of an elongation factor (SelB) responsible for delivering the mature Sec-tRNA^Sec into the ribosome by the interaction with the Selenocysteine Insertion Sequence (SECIS). Besides this mechanism being already described, the sequential events for Sec-tRNA^Sec and SECIS specific recognition remain unclear. In this study, we determined the order of events of the interactions between the proteins and RNAs involved in Sec incorporation. Dissociation constants between SelB and the native as well as unacylated-tRNA^Sec variants demonstrated that the acceptor stem and variable arm are essential for SelB recognition. Moreover, our data support the sequence of molecular events where GTP-activated SelB strongly interacts with SelA.tRNA^Sec. Subsequently, SelB.GTP.tRNA^Sec recognizes the mRNA SECIS to deliver the tRNA^Sec to the ribosome. SelB in complex with its specific RNAs were examined using Hydrogen/Deuterium exchange mapping that allowed the determination of the molecular envelopes and its secondary structural variations during the complex assembly. Our results demonstrate the ordering of events in Sec incorporation and contribute to the full comprehension of the tRNA^Sec role in the Sec amino acid biosynthesis, as well as extending the knowledge of synthetic biology and the expansion of the genetic code.     

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.     

Tertiary structure of bacterial selenocysteine tRNA

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA^Sec) is first ligated with serine by seryl-tRNA synthetase (SerRS). In the present study, we determined the 3.1 Å crystal structure of the tRNA^Sec from the bacterium Aquifex aeolicus, in complex with the heterologous SerRS from the archaeon Methanopyrus kandleri. The bacterial tRNA^Sec assumes the L-shaped structure, from which the long extra arm protrudes. Although the D-arm conformation and the extra-arm orientation are similar to those of eukaryal/archaeal tRNA^Secs, A. aeolicus tRNA^Sec has unique base triples, G14:C21:U8 and C15:G20a:G48, which occupy the positions corresponding to the U8:A14 and R15:Y48 tertiary base pairs of canonical tRNAs. Methanopyrus kandleri SerRS exhibited serine ligation activity toward A. aeolicus tRNA^Sec in vitro. The SerRS N-terminal domain interacts with the extra-arm stem and the outer corner of tRNA^Sec. Similar interactions exist in the reported tRNA^Ser and SerRS complex structure from the bacterium Thermus thermophilus. Although the catalytic C-terminal domain of M. kandleri SerRS lacks interactions with A. aeolicus tRNA^Sec in the present complex structure, the conformational flexibility of SerRS is likely to allow the CCA terminal region of tRNA^Sec to enter the SerRS catalytic site.     

Translational Redefinition of UGA Codons Is Regulated by Selenium Availability

Incorporation of selenium into ∼25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA^[Ser]Sec Um34 methylation. Furthermore, we find evidence for translation in the 5′-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins.