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Chlamydomonas reinhardtii selenocysteine tRNA[Ser]Sec.

ABSTRACT: Eukaryotic selenocysteine (Sec) protein insertion machinery was thought to be restricted to animals, but the occurrence of both Sec-containing proteins and the Sec insertion system was recently found in Chlamydomonas reinhardtii, a member of the plant kingdom. Herein, we used RT-PCR to determine the sequence of C. reinhardtii Sec tRNA[Ser]Sec, the first non-animal eukaryotic Sec tRNA[Ser]Sec sequence. Like its animal counterpart, it is 90 nucleotides in length, is aminoacylated with serine by seryl-tRNA synthetase, and decodes specifically UGA. Evolutionary analyses of known Sec tRNAs identify the C. reinhardtii form as the most diverged eukaryotic Sec tRNA[Ser]Sec and reveal a common origin for this tRNA in bacteria, archaea, and eukaryotes.

SUBMITTER: Rao M

PROVIDER: S-EPMC1370458 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Chlamydomonas reinhardtii selenocysteine tRNA[Ser]Sec.

Rao Mahadev M Carlson Bradley A BA Novoselov Sergey V SV Weeks Donald P DP Gladyshev Vadim N VN Hatfield Dolph L DL

RNA (New York, N.Y.) 20030801 8

Eukaryotic selenocysteine (Sec) protein insertion machinery was thought to be restricted to animals, but the occurrence of both Sec-containing proteins and the Sec insertion system was recently found in Chlamydomonas reinhardtii, a member of the plant kingdom. Herein, we used RT-PCR to determine the sequence of C. reinhardtii Sec tRNA[Ser]Sec, the first non-animal eukaryotic Sec tRNA[Ser]Sec sequence. Like its animal counterpart, it is 90 nucleotides in length, is aminoacylated with serine by se ...[more]

PMID: 12869703

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Inhibition of selenocysteine tRNA[Ser]Sec aminoacylation provides evidence that aminoacylation is required for regulatory methylation of this tRNA.

Project description:There are two isoforms of selenocysteine (Sec) tRNA([Ser]Sec) that differ by a single methyl group, Um34. The non-Um34 isoform supports the synthesis of a subclass of selenoproteins, designated housekeeping, while the Um34 isoform supports the expression of another subclass, designated stress-related selenoproteins. Herein, we investigated the relationship between tRNA([Ser]Sec) aminoacylation and Um34 synthesis which is the last step in the maturation of this tRNA. Mutation of the discriminator base at position 73 in tRNA([Ser]Sec) dramatically reduced aminoacylation with serine, as did an inhibitor of seryl-tRNA synthetase, SB-217452. Although both the mutation and the inhibitor prevented Um34 synthesis, neither precluded the synthesis of any other of the known base modifications on tRNA([Ser]Sec) following microinjection and incubation of the mutant tRNA([Ser]Sec) transcript, or the wild type transcript along with inhibitor, in Xenopus oocytes. The data demonstrate that Sec tRNA([Ser]Sec) must be aminoacylated for Um34 addition. The fact that selenium is required for Um34 methylation suggests that Sec must be attached to its tRNA for Um34 methylation. This would explain why selenium is essential for the function of Um34 methylase and provides further insights into the hierarchy of selenoprotein expression.

| S-EPMC3124823 | biostudies-literature

A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA[Ser]Sec Gene.

Project description:The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3' UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA[Ser]Sec). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA[Ser]Sec and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA[Ser]Sec gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA[Ser]Sec and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism.

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A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis.

Project description:Selenium is biologically active through the functions of selenoproteins that contain the amino acid selenocysteine. This amino acid is translated in response to in-frame UGA codons in mRNAs that include a SECIS element in its 3' untranslated region, and this process requires a unique tRNA, referred to as tRNA([Ser]Sec). The translation of UGA as selenocysteine, rather than its use as a termination signal, is a candidate restriction point for the regulation of selenoprotein synthesis by selenium. A specialized reporter construct was used that permits the evaluation of SECIS-directed UGA translation to examine mechanisms of the regulation of selenoprotein translation. Using SECIS elements from five different selenoprotein mRNAs, UGA translation was quantified in response to selenium supplementation and alterations in tRNA([Ser]Sec) levels and isoform distributions. Although each of the evaluated SECIS elements exhibited differences in their baseline activities, each was stimulated to a similar extent by increased selenium or tRNA([Ser]Sec) levels and was inhibited by diminished levels of the methylated isoform of tRNA([Ser]Sec) achieved using a dominant-negative acting mutant tRNA([Ser]Sec). tRNA([Ser]Sec) was found to be limiting for UGA translation under conditions of high selenoprotein mRNA in both a transient reporter assay and in cells with elevated GPx-1 mRNA. This and data indicating increased amounts of the methylated isoform of tRNA([Ser]Sec) during selenoprotein translation indicate that it is this isoform that is translationally active and that selenium-induced tRNA methylation is a mechanism of regulation of the synthesis of selenoproteins.

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Identification and characterization of phosphoseryl-tRNA[Ser]Sec kinase.

Project description:In 1970, a kinase activity that phosphorylated a minor species of seryl-tRNA to form phosphoseryl-tRNA was found in rooster liver [Maenpaa, P. H. & Bernfield, M. R. (1970) Proc. Natl. Acad. Sci. USA 67, 688-695], and a minor seryl-tRNA that decoded the nonsense UGA was detected in bovine liver. The phosphoseryl-tRNA and the minor UGA-decoding seryl-tRNA were subsequently identified as selenocysteine (Sec) tRNA[Ser]Sec, but the kinase activity remained elusive. Herein, by using a comparative genomics approach that searched completely sequenced archaeal genomes for a kinase-like protein with a pattern of occurrence similar to that of components of Sec insertion machinery, we detected a candidate gene for mammalian phosphoseryl-tRNA[Ser]Sec kinase (pstk). Mouse pstk was cloned, and the gene product (PSTK) was expressed and characterized. PSTK specifically phosphorylated the seryl moiety on seryl-tRNA[Ser]Sec and, in addition, had a requirement for ATP and Mg2+. Proteins with homology to mammalian PSTK occur in Drosophila, Caenorhabditis elegans, Methanopyrus kandleri, and Methanococcus jannaschii, suggesting a conservation of its function across archaea and eukaryotes that synthesize selenoproteins and the absence of this function in bacteria, plants, and yeast. The fact that PSTK has been highly conserved in evolution suggests that it plays an important role in selenoprotein biosynthesis and/or regulation.

| S-EPMC516484 | biostudies-literature

Decameric SelA•tRNA(Sec) ring structure reveals mechanism of bacterial selenocysteine formation.

Project description:The 21st amino acid, selenocysteine (Sec), is synthesized on its cognate transfer RNA (tRNA(Sec)). In bacteria, SelA synthesizes Sec from Ser-tRNA(Sec), whereas in archaea and eukaryotes SepSecS forms Sec from phosphoserine (Sep) acylated to tRNA(Sec). We determined the crystal structures of Aquifex aeolicus SelA complexes, which revealed a ring-shaped homodecamer that binds 10 tRNA(Sec) molecules, each interacting with four SelA subunits. The SelA N-terminal domain binds the tRNA(Sec)-specific D-arm structure, thereby discriminating Ser-tRNA(Sec) from Ser-tRNA(Ser). A large cleft is created between two subunits and accommodates the 3'-terminal region of Ser-tRNA(Sec). The SelA structures together with in vivo and in vitro enzyme assays show decamerization to be essential for SelA function. SelA catalyzes pyridoxal 5'-phosphate-dependent Sec formation involving Arg residues nonhomologous to those in SepSecS. Different protein architecture and substrate coordination of the bacterial enzyme provide structural evidence for independent evolution of the two Sec synthesis systems present in nature.

| S-EPMC3976565 | biostudies-literature

The Effect of tRNA[Ser]Sec Isopentenylation on Selenoprotein Expression.

Project description:Transfer RNA[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U34 to mcm5Um occurs independently of isopentenylation of A37 in tRNA[Ser]Sec. Western blotting and 75Se metabolic labeling showed only moderate effects on selenoprotein levels and 75Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2'O-methylation of U34 in tRNA[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.

| S-EPMC8583801 | biostudies-literature

Using selenocysteine-specific reporters to screen for efficient tRNASec variants.

Project description:The unique properties of selenocysteine (Sec) have generated an interest in the scientific community to site-specifically incorporate Sec into a protein of choice. Current technologies have rewired the natural Sec-specific translation factor-dependent selenoprotein biosynthesis pathway by harnessing the canonical elongation factor (EF-Tu) to simplify the requirements for Sec incorporation in Escherichia coli. This strategy is versatile and can be applied to Sec incorporation at any position in a protein of interest. However, selenoprotein production is still limited by yield and serine misincorporation. This protocol outlines a method in E. coli to design and optimize tRNA libraries which can be selected and screened for by the use of Sec-specific intein-based reporters. This provides a fast and simple way to engineer tRNAs with enhanced Sec-incorporation ability.

| S-EPMC8841065 | biostudies-literature

Selenoproteins and selenocysteine insertion system in the model plant cell system, Chlamydomonas reinhardtii.

Project description:Known eukaryotic selenocysteine (Sec)-containing proteins are animal proteins, whereas selenoproteins have not been found in yeast and plants. Surprisingly, we detected selenoproteins in a member of the plant kingdom, Chlamydomonas reinhardtii, and directly identified two of them as phospholipid hydroperoxide glutathione peroxidase and selenoprotein W homologs. Moreover, a selenocysteyl-tRNA was isolated that recognized specifically the Sec codon UGA. Subsequent gene cloning and bioinformatics analyses identified eight additional selenoproteins, including methionine-S-sulfoxide reductase, a selenoprotein specific to Chlamydomonas: Chlamydomonas selenoprotein genes contained selenocysteine insertion sequence (SECIS) elements that were similar, but not identical, to those of animals. These SECIS elements could direct selenoprotein synthesis in mammalian cells, indicating a common origin of plant and animal Sec insertion systems. We found that selenium is required for optimal growth of Chlamydomonas: Finally, evolutionary analyses suggested that selenoproteins present in Chlamydomonas and animals evolved early, and were independently lost in land plants, yeast and some animals.

| S-EPMC126117 | biostudies-literature

Construction of anti-codon table of the plant kingdom and evolution of tRNA selenocysteine (tRNASec).

Project description:BackgroundThe tRNAs act as a bridge between the coding mRNA and incoming amino acids during protein translation. The anti-codon of tRNA recognizes the codon of the mRNA and deliver the amino acid into the protein translation chain. However, we did not know about the exact abundance of anti-codons in the genome and whether the frequency of abundance remains same across the plant lineage or not.ResultsTherefore, we analysed the tRNAnome of 128 plant species and reported an anti-codon table of the plant kingdom. We found that CAU anti-codon of tRNAMet has highest (5.039%) whereas GCG anti-codon of tRNAArg has lowest (0.004%) abundance. However, when we compared the anti-codon frequencies according to the tRNA isotypes, we found tRNALeu (7.808%) has highest abundance followed by tRNASer (7.668%) and tRNAGly (7.523%). Similarly, suppressor tRNA (0.036%) has lowest abundance followed by tRNASec (0.066%) and tRNAHis (2.109). The genome of Ipomoea nil, Papaver somniferum, and Zea mays encoded the highest number of anti-codons (isoacceptor) at 59 each whereas the genome of Ostreococcus tauri was found to encode only 18 isoacceptors. The tRNASec genes undergone losses more frequently than duplication and we found that tRNASec showed anti-codon switch during the course of evolution.ConclusionThe anti-codon table of the plant tRNA will enable us to understand the synonymous codon usage of the plant kingdom and can be very helpful to understand which codon is preferred over other during the translation.

| S-EPMC7678280 | biostudies-literature

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