RESUMEN
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.
Asunto(s)
ARN de Transferencia Aminoácido-Específico , Selenocisteína , Escherichia coli/genética , Escherichia coli/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia Aminoácido-Específico/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismoRESUMEN
Objective: Graves' disease (GD) related hyperthyroidism (HT) has profound effects on metabolic activity and metabolism of macromolecules affecting energy homeostasis. In this study, we aimed to get a comprehensive understanding of the metabolic changes and their clinical relevance in GD children. Methods: We investigated serum substances from 30 newly diagnosed GD children and 30 age- and gender-matched healthy controls. We explored the metabolomics using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) analysis, and then analyzed the metabolomic data via multivariate statistical analysis. Results: By untargeted metabolomic analysis, a total of 730 metabolites were identified in all participants, among which 48 differential metabolites between GD and control groups were filtered out, including amino acids, dipeptides, lipids, purines, etc. Among these metabolites, 33 were detected with higher levels, while 15 with lower levels in GD group compared to controls. Pathway analysis showed that HT had a significant impact on aminoacyl-transfer ribonucleic acid (tRNA) biosynthesis, several amino acids metabolism, purine metabolism, and pyrimidine metabolism. Conclusion: In this study, via untargeted metabolomics analysis, significant variations of serum metabolomic patterns were detected in GD children.
Asunto(s)
Enfermedad de Graves/metabolismo , Metabolómica , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Biomarcadores , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Femenino , Enfermedad de Graves/genética , Humanos , Masculino , Redes y Vías Metabólicas , Purinas/metabolismo , Pirimidinas/metabolismo , ARN de Transferencia Aminoácido-Específico/biosíntesis , ARN de Transferencia Aminoácido-Específico/genética , Espectrometría de Masas en TándemRESUMEN
Selenium, a trace element fundamental to human health, is incorporated as the amino acid selenocysteine (Sec) into more than 25 proteins, referred to as selenoproteins. Human mutations in SECISBP2, SEPSECS and TRU-TCA1-1, three genes essential in the selenocysteine incorporation pathway, affect the expression of most if not all selenoproteins. Systemic selenoprotein deficiency results in a complex, multifactorial disorder, reflecting loss of selenoprotein function in specific tissues and/or long-term impaired selenoenzyme-mediated defence against oxidative and endoplasmic reticulum stress. SEPSECS mutations are associated with a predominantly neurological phenotype with progressive cerebello-cerebral atrophy. Selenoprotein deficiency due to SECISBP2 and TRU-TCA1-1 defects are characterized by abnormal circulating thyroid hormones due to lack of Sec-containing deiodinases, low serum selenium levels (low SELENOP, GPX3), with additional features (myopathy due to low SELENON; photosensitivity, hearing loss, increased adipose mass and function due to reduced antioxidant and endoplasmic reticulum stress defence) in SECISBP2 cases. Antioxidant therapy ameliorates oxidative damage in cells and tissues of patients, but its longer term benefits remain undefined. Ongoing surveillance of patients enables ascertainment of additional phenotypes which may provide further insights into the role of selenoproteins in human biological processes.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Aminoacil-ARNt Sintetasas/genética , ARN de Transferencia Aminoácido-Específico/genética , Proteínas de Unión al ARN/genética , Selenoproteínas/deficiencia , Humanos , MutaciónRESUMEN
The role of the essential trace element selenium in hypothalamic physiology has begun to come to light over recent years. Selenium is used to synthesize a family of proteins participating in redox reactions called selenoproteins, which contain a selenocysteine residue in place of a cysteine. Past studies have shown that disrupted selenoprotein expression in the hypothalamus can adversely impact energy homeostasis. There is also evidence that selenium supports leptin signaling in the hypothalamus by maintaining proper redox balance. In this study, we generated mice with conditional knockout of the selenocysteine tRNA[Ser]Sec gene (Trsp) in an orexigenic cell population called agouti-related peptide (Agrp)-positive neurons. We found that female TrspAgrpKO mice gain less weight while on a high-fat diet, which occurs due to changes in adipose tissue activity. Female TrspAgrpKO mice also retained hypothalamic sensitivity to leptin administration. Male mice were unaffected, however, highlighting the sexually dimorphic influence of selenium on neurobiology and energy homeostasis. These findings provide novel insight into the role of selenoproteins within a small yet heavily influential population of hypothalamic neurons.
Asunto(s)
Peso Corporal/efectos de los fármacos , Dieta Alta en Grasa , Leptina/farmacología , Neuronas/metabolismo , ARN de Transferencia Aminoácido-Específico/genética , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/patología , Animales , Dióxido de Carbono/metabolismo , Metabolismo Energético , Femenino , Prueba de Tolerancia a la Glucosa , Leptina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/metabolismo , Obesidad/patología , Obesidad/veterinaria , ARN de Transferencia Aminoácido-Específico/metabolismo , Transducción de SeñalRESUMEN
In bacteria, selenocysteine (Sec) is incorporated into proteins via the recoding of a particular codon, the UGA stop codon in most cases. Sec-tRNASec is delivered to the ribosome by the Sec-dedicated elongation factor SelB that also recognizes a Sec-insertion sequence element following the codon on the mRNA. Since the excess of SelB may lead to sequestration of Sec-tRNASec under selenium deficiency or oxidative stress, the expression levels of SelB and tRNASec should be regulated. In this bioinformatic study, I analyzed the Rhizobiales SelB species because they were annotated to have a non-canonical C-terminal extension. I found that the open reading frame (ORF) of diverse Alphaproteobacteria selB genes includes an entire tRNASec sequence (selC) and overlaps with the start codon of the downstream ORF. A remnant tRNASec sequence was found in the Sinorhizobium melilotiselB genes whose products have a shorter C-terminal extension. Similar overlapping traits were found in Gammaproteobacteria and Nitrospirae. I hypothesized that once the tRNASec moiety is folded and processed, the expression of the full-length SelB may be repressed. This is the first report on a nested tRNA gene inside a protein ORF in bacteria.
Asunto(s)
Alphaproteobacteria/genética , Proteínas Bacterianas/genética , Selenocisteína/genética , Proteínas Bacterianas/metabolismo , Codón de Terminación/metabolismo , Biología Computacional/métodos , Conformación de Ácido Nucleico , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN de Transferencia/genética , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia Aminoácido-Específico/metabolismo , Ribosomas/metabolismo , Selenocisteína/metabolismoRESUMEN
Selenoproteins are a class of proteins with the selenium-containing amino acid selenocysteine (Sec) in their primary structure. Sec is incorporated into selenoproteins via recoding of the stop codon UGA, with specific cis and trans factors required during translation to avoid UGA recognition as a stop codon, including a Sec-specific tRNA, tRNA[Ser]Sec, encoded in mice by the gene Trsp. Whole-body deletion of Trsp in mouse is embryonically lethal, while targeted deletion of Trsp in mice has been used to understand the role of selenoproteins in the health and physiology of various tissues. We developed a mouse model with the targeted deletion of Trsp in brown adipocytes (Trspf/f-Ucp1-Cre+/-), a cell type predominant in brown adipose tissue (BAT) controlling energy expenditure via activation of adaptive thermogenesis, mostly using uncoupling protein 1 (Ucp1). At room temperature, Trspf/f-Ucp1-Cre+/- mice maintain oxygen consumption and Ucp1 expression, with male Trspf/f-Ucp1-Cre+/- mice accumulating more triglycerides in BAT than both female Trspf/f-Ucp1-Cre+/- mice or Trspf/f controls. Acute cold exposure neither reduced core body temperature nor changed the expression of selenoprotein iodothyronine deiodinase type II (Dio2), a marker of adaptive thermogenesis, in Trspf/f-Ucp1-Cre+/- mice. Microarray analysis of BAT from Trspf/f-Ucp1-Cre+/- mice revealed glutathione S-transferase alpha 3 (Gsta3) and ELMO domain containing 2 (Elmod2) as the transcripts most affected by the loss of Trsp. Male Trspf/f-Ucp1-Cre+/- mice showed mild hypothyroidism while downregulating thyroid hormone-responsive genes Thrsp and Tshr in their BATs. In summary, modest changes in the BAT of Trspf/f-Ucp1-Cre +/- mice implicate a mild thyroid hormone dysfunction in brown adipocytes.
Asunto(s)
Adipocitos Marrones/metabolismo , Selenoproteínas/metabolismo , Termogénesis , Tejido Adiposo Pardo/metabolismo , Animales , Vías Biosintéticas , Células Cultivadas , Respuesta al Choque por Frío , Metabolismo Energético , Femenino , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , ARN de Transferencia Aminoácido-Específico/genética , Proteína Desacopladora 1/genéticaRESUMEN
Increasing evidence demonstrates that tRNA-derived fragments (tRFs) exert important effects and are dysregulated in various human cancer types. However, their roles in gastric cancer (GC) remain unknown. Here we identified the functional effects of tRF-3019a (derived from tRNA-Ala-AGC-1-1) in GC. We demonstrated that tRF-3019a was upregulated in GC tissues and cell lines. Phenotypic studies revealed that tRF-3019a overexpression enhances GC cell proliferation, migration and invasion. Conversely, tRF-3019a knockdown inhibits GC cell malignant activities. Mechanistic investigation implies that tRF-3019a directly regulates tumor suppressor gene FBXO47. Furthermore, tRF-3019a levels may discriminate GC tissues from nontumorous tissues. Taken together, our results reveal that tRF-3019a modulates GC cell proliferation, migration and invasion by targeting FBXO47, and it may serve as a potential diagnostic biomarker for GC.
Asunto(s)
ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia/metabolismo , Neoplasias Gástricas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Bases , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Conformación de Ácido Nucleico , TransfecciónRESUMEN
tRNA synthetases are responsible for decoding the molecular information, from codons to amino acids. Seryl-tRNA synthetase (SerRS), besides the five isoacceptors of tRNASer, recognizes tRNA[Ser]Sec for the incorporation of selenocysteine (Sec, U) into selenoproteins. The selenocysteine synthesis pathway is known and is dependent on several protein-protein and protein-RNA interactions. Those interactions are not fully described, in particular, involving tRNA[Ser]Sec and SerRS. Here we describe the molecular interactions between the Escherichia coli Seryl-tRNA synthetase (EcSerRS) and tRNA[Ser]Sec in order to determine their specificity, selectivity and binding order, leading to tRNA aminoacylation. The dissociation constant of EcSerRS and tRNA[Ser]Sec was determined as (126 ± 20) nM. We also demonstrate that EcSerRS binds initially to tRNA[Ser]Sec in the presence of ATP for further recognition by E. coli selenocysteine synthetase (EcSelA) for Ser to Sec conversion. The proposed studies clarify the mechanism of tRNA[Ser]Sec incorporation in Bacteria as well as of other domains of life.
Asunto(s)
Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN de Transferencia Aminoácido-Específico/metabolismo , ARN de Transferencia de Cisteína/metabolismo , Serina-ARNt Ligasa/metabolismo , Transferasas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Escherichia coli/genética , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia de Cisteína/genética , Serina-ARNt Ligasa/genética , Termodinámica , Aminoacilación de ARN de Transferencia/genética , Transferasas/genéticaRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas/genética , Codón de Terminación/genética , ARN de Transferencia Aminoácido-Específico/genética , Ribosomas/metabolismo , Selenocisteína/metabolismo , Virión/metabolismo , Secuencia de Bases , Edición Génica , Células HEK293 , Células HeLa , Humanos , Mutación INDEL/genética , Conformación de Ácido Nucleico , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia Aminoácido-Específico/química , Selenio/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismoRESUMEN
Selenocysteine (Sec) lacks a cognate aminoacyl-tRNA synthetase. Instead, seryl-tRNA synthetase (SerRS) produces Ser-tRNASec , which is subsequently converted by selenocysteine synthase to Sec-tRNASec . Escherichia coli SerRS serylates tRNASec poorly; this may hinder efficient production of designer selenoproteins in vivo. Guided by structural modelling and selection for chloramphenicol acetyltransferase activity, we evolved three SerRS variants capable of improved Ser-tRNASec synthesis. They display 10-, 8-, and 4-fold increased kcat /KM values compared to wild-type SerRS using synthetic tRNASec species as substrates. The enzyme variants also facilitate in vivo read-through of a UAG codon in the position of the critical serine146 of chloramphenicol acetyltransferase. These results indicate that the naturally evolved SerRS is capable of further evolution for increased recognition of a specific tRNA isoacceptor.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia de Serina/genética , Serina-ARNt Ligasa/genética , Secuencia de Bases , Codón de Terminación/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Dominios Proteicos , ARN de Transferencia Aminoácido-Específico/química , ARN de Transferencia Aminoácido-Específico/metabolismo , ARN de Transferencia de Serina/química , ARN de Transferencia de Serina/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Serina/genética , Serina/metabolismo , Serina-ARNt Ligasa/química , Serina-ARNt Ligasa/metabolismo , Especificidad por SustratoRESUMEN
Genetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded noncanonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei ( Mma PylRS/PylT) being the most active and versatile to date. We found a pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that this PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its noncognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Euryarchaeota/enzimología , ARN de Transferencia Aminoácido-Específico/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Sitios de Unión , Codón de Terminación/genética , Células HEK293 , Humanos , Lisina/análogos & derivados , Lisina/genética , Mutación , Ingeniería de Proteínas/métodos , ARN de Transferencia Aminoácido-Específico/genética , Especificidad por SustratoRESUMEN
The selenocysteine (Sec) tRNA[Ser]Sec population consists of two isoforms that differ from each other by a single 2'-O-methylribosyl moiety at position 34 (Um34). These two isoforms, which are encoded in a single gene, Trsp, and modified posttranscriptionally, are involved individually in the synthesis of two subclasses of selenoproteins, designated housekeeping and stress-related selenoproteins. Techniques used in obtaining these isoforms for their characterization include extraction of RNA from mammalian cells and tissues, purifying the tRNA[Ser]Sec population by one or more procedures, and finally resolving the two isoforms from each other. Since some of the older techniques for isolating tRNA[Ser]Sec and resolving the isoforms are used in only a few laboratories, these procedures will be discussed briefly and references provided for more detailed information, while the more recently developed procedures are discussed in detail. In addition, a novel technique that was developed in sequencing tRNA[Ser]Sec for identifying their occurrence in other organisms is also presented.
Asunto(s)
ARN de Transferencia Aminoácido-Específico/genética , Selenoproteínas/genética , Animales , Northern Blotting , Cromatografía de Afinidad , Cromatografía de Fase Inversa , Humanos , Marcaje Isotópico , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN de Transferencia Aminoácido-Específico/química , Radioisótopos de Selenio , Selenoproteínas/química , Selenoproteínas/aislamiento & purificación , Análisis de Secuencia de ARNRESUMEN
RNA polymerase III catalyses the synthesis of tRNAs in eukaryotic organisms. Through combined biochemical and structural characterisation, multiple auxiliary factors have been identified alongside RNA Polymerase III as critical in both facilitating and regulating transcription. Together, this machinery forms dynamic multi-protein complexes at tRNA genes which are required for polymerase recruitment, DNA opening and initiation and elongation of the tRNA transcripts. Central to the function of these complexes is their ability to undergo multiple conformational changes and rearrangements that regulate each step. Here, we discuss the available biochemical and structural data on the structural plasticity of multi-protein complexes involved in RNA Polymerase III transcriptional initiation and facilitated re-initiation during tRNA synthesis. Increasingly, structural information is becoming available for RNA polymerase III and its functional complexes, allowing for a deeper understanding of tRNA transcriptional initiation. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.
Asunto(s)
ARN Polimerasa III/metabolismo , ARN de Transferencia/biosíntesis , Iniciación de la Transcripción Genética , Animales , Células Eucariotas/metabolismo , Humanos , Modelos Genéticos , Complejos Multiproteicos/metabolismo , Regiones Promotoras Genéticas/genética , Subunidades de Proteína , ARN Polimerasa III/química , ARN de Transferencia/genética , ARN de Transferencia Aminoácido-Específico/biosíntesis , ARN de Transferencia Aminoácido-Específico/genética , Elongación de la Transcripción Genética , Factores de Transcripción/genéticaRESUMEN
Seryl-tRNA synthetase (SerRS) attaches L-serine to the cognate serine tRNA (tRNASer) and the noncognate selenocysteine tRNA (tRNASec). The latter activity initiates the anabolic cycle of selenocysteine (Sec), proper decoding of an in-frame Sec UGA codon, and synthesis of selenoproteins across all domains of life. While the accuracy of SerRS is important for overall proteome integrity, it is its substrate promiscuity that is vital for the integrity of the selenoproteome. This raises a question as to what elements in the two tRNA species, harboring different anticodon sequences and adopting distinct folds, facilitate aminoacylation by a common aminoacyl-tRNA synthetase. We sought to answer this question by analyzing the ability of human cytosolic SerRS to bind and act on tRNASer, tRNASec, and 10 mutant and chimeric constructs in which elements of tRNASer were transposed onto tRNASec We show that human SerRS only subtly prefers tRNASer to tRNASec, and that discrimination occurs at the level of the serylation reaction. Surprisingly, the tRNA mutants predicted to adopt either the 7/5 or 8/5 fold are poor SerRS substrates. In contrast, shortening of the acceptor arm of tRNASec by a single base pair yields an improved SerRS substrate that adopts an 8/4 fold. We suggest that an optimal tertiary arrangement of structural elements within tRNASec and tRNASer dictate their utility for serylation. We also speculate that the extended acceptor-TΨC arm of tRNASec evolved as a compromise for productive binding to SerRS while remaining the major recognition element for other enzymes involved in Sec and selenoprotein synthesis.
Asunto(s)
ARN de Transferencia Aminoácido-Específico/metabolismo , ARN de Transferencia de Serina/metabolismo , Serina-ARNt Ligasa/metabolismo , Secuencia de Bases , Sitios de Unión , Citosol/enzimología , Humanos , Cinética , Modelos Moleculares , Mutagénesis , Conformación de Ácido Nucleico , Pliegue del ARN , ARN de Transferencia Aminoácido-Específico/química , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia de Serina/química , ARN de Transferencia de Serina/genética , Especificidad por SustratoRESUMEN
Selenocysteine (Sec) is known as the 21st amino acid, a cysteine analogue with selenium replacing sulphur. Sec is inserted co-translationally in a small fraction of proteins called selenoproteins. In selenoprotein genes, the Sec specific tRNA (tRNASec) drives the recoding of highly specific UGA codons from stop signals to Sec. Although found in organisms from the three domains of life, Sec is not universal. Many species are completely devoid of selenoprotein genes and lack the ability to synthesize Sec. Since tRNASec is a key component in selenoprotein biosynthesis, its efficient identification in genomes is instrumental to characterize the utilization of Sec across lineages. Available tRNA prediction methods fail to accurately predict tRNASec, due to its unusual structural fold. Here, we present Secmarker, a method based on manually curated covariance models capturing the specific tRNASec structure in archaea, bacteria and eukaryotes. We exploited the non-universality of Sec to build a proper benchmark set for tRNASec predictions, which is not possible for the predictions of other tRNAs. We show that Secmarker greatly improves the accuracy of previously existing methods constituting a valuable tool to identify tRNASec genes, and to efficiently determine whether a genome contains selenoproteins. We used Secmarker to analyze a large set of fully sequenced genomes, and the results revealed new insights in the biology of tRNASec, led to the discovery of a novel bacterial selenoprotein family, and shed additional light on the phylogenetic distribution of selenoprotein containing genomes. Secmarker is freely accessible for download, or online analysis through a web server at http://secmarker.crg.cat.
Asunto(s)
Mapeo Cromosómico/métodos , Marcadores Genéticos/genética , Genoma/genética , Ensayos Analíticos de Alto Rendimiento/métodos , ARN de Transferencia Aminoácido-Específico/genética , Aminoacil-ARN de Transferencia/genética , Algoritmos , Componentes Genómicos/genética , SelenocisteínaRESUMEN
Dual-assignment of codons as termination and elongation codons is used to expand the genetic code. In mammals, UGA can be reassigned to selenocysteine during translation of selenoproteins by a mechanism involving a 3Î untranslated region (UTR) selenocysteine insertion sequence (SECIS) and the SECIS-binding protein Secisbp2. Here, we present data from ribosome profiling, RNA-Seq and mRNA half-life measurements that support distinct roles for Secisbp2 in UGA-redefinition and mRNA stability. Conditional deletions of the Secisbp2 and Trsp (tRNASec) genes in mouse liver were compared to determine if the effects of Secisbp2 loss on selenoprotein synthesis could be attributed entirely to the inability to incorporate Sec. As expected, tRNASec depletion resulted in loss of ribosome density downstream of all UGA-Sec codons. In contrast, the absence of Secisbp2 resulted in variable effects on ribosome density downstream of UGA-Sec codons that demonstrate gene-specific differences in Sec incorporation. For several selenoproteins in which loss of Secisbp2 resulted in greatly diminished mRNA levels, translational activity and Sec incorporation efficiency were shown to be unaffected on the remaining RNA. Collectively, these results demonstrate that Secisbp2 is not strictly required for Sec incorporation and has a distinct role in stabilizing mRNAs that can be separated from its effects on UGA-redefinition.
Asunto(s)
Codón de Terminación , Estabilidad del ARN , ARN Mensajero/metabolismo , ARN de Transferencia Aminoácido-Específico/genética , Proteínas de Unión al ARN/fisiología , Selenoproteínas/genética , Animales , Células Cultivadas , Hepatocitos/metabolismo , Masculino , Metilación , Ratones , Ratones Noqueados , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , ARN de Transferencia Aminoácido-Específico/metabolismo , Proteínas de Unión al ARN/genética , Ribosomas/metabolismo , Selenoproteínas/biosíntesisRESUMEN
The covalent coupling of cognate amino acid-tRNA pairs by corresponding aminoacyl-tRNA synthetases (aaRS) defines the genetic code and provides aminoacylated tRNAs for ribosomal protein synthesis. Besides the cognate substrate, some non-cognate amino acids may also compete for tRNA aminoacylation. However, their participation in protein synthesis is generally prevented by an aaRS proofreading activity located in the synthetic site and in a separate editing domain. These mechanisms, coupled with the ability of certain aaRSs to discriminate well against non-cognate amino acids in the synthetic reaction alone, define the accuracy of the aminoacylation reaction. aaRS quality control may also act as a gatekeeper for the standard genetic code and prevents infiltration by natural amino acids that are not normally coded for protein biosynthesis. This latter finding has reinforced interest in understanding the principles that govern discrimination against a range of potential non-cognate amino acids. This paper presents an overview of the kinetic assays that have been established for monitoring synthetic and editing reactions with cognate and non-cognate amino acid substrates. Taking into account the peculiarities of non-cognate reactions, the specific controls needed and the dedicated experimental designs are discussed in detail. Kinetic partitioning within the synthetic and editing sites controls the balance between editing and aminoacylation. We describe in detail steady-state and single-turnover approaches for the analysis of synthetic and editing reactions, which ultimately enable mechanisms of amino acid discrimination to be determined.
Asunto(s)
Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Pruebas de Enzimas , Edición de ARN , ARN de Transferencia Aminoácido-Específico/genética , Aminoacilación de ARN de Transferencia , Adenosina Trifosfato/metabolismo , Aminoacil-ARNt Sintetasas/genética , Código Genético , Hidrólisis , Cinética , ARN de Transferencia Aminoácido-Específico/metabolismo , Especificidad por SustratoRESUMEN
The fidelity of tRNA aminoacylation is a critical determinant for the ultimate accuracy of protein synthesis. Although aminoacyl-tRNA synthetases are assumed to consistently maintain high tRNA charging fidelity, recent evidence has demonstrated that the fidelity of the aminoacylation reaction can be actively regulated and liable to change. Accordingly, the ability to conveniently assay the fidelity of tRNA charging is becoming increasingly relevant for studying mistranslation. Here we describe a combined radioactivity and microarray based method that can quantitatively elucidate which individual cognate or noncognate tRNA isoacceptors are charged with amino acid. In this technique, in vitro tRNA charging reactions or in vivo pulse-labeling is performed using a radiolabeled amino acid and tRNA microarrays are used to distinguish tRNA isoacceptors in total tRNA. During the tRNA array hybridization, each tRNA will hybridize to its unique probe and subsequent phosphorimaging of the array can determine which tRNAs were aminoacylated with the radiolabeled amino acid. The method can be used to assess the fidelity of tRNA charging in vivo or in vitro and can be applied to any organism with annotated tRNA genes.
Asunto(s)
Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Análisis por Micromatrices/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , ARN de Transferencia Aminoácido-Específico/genética , Aminoacilación de ARN de Transferencia , Aminoacil-ARNt Sintetasas/genética , Radioisótopos de Carbono , Escherichia coli/enzimología , Escherichia coli/genética , Impresión/métodos , Sondas ARN/síntesis química , ARN de Transferencia Aminoácido-Específico/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Radioisótopos de Azufre , TritioRESUMEN
In addition to their role in correctly attaching specific amino acids to cognate tRNAs, aminoacyl-tRNA synthetases (aaRS) have been found to possess many alternative functions and often bind to and act on other nucleic acids. In contrast to the well-defined 3D structure of tRNA, the structures of many of the other RNAs recognized by aaRSs have not been solved. Despite advances in the use of X-ray crystallography (XRC), nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) for structural characterization of biomolecules, significant challenges to solving RNA structures still exist. Recently, small-angle X-ray scattering (SAXS) has been increasingly employed to characterize the 3D structures of RNAs and RNA-protein complexes. SAXS is capable of providing low-resolution tertiary structure information under physiological conditions and with less intensive sample preparation and data analysis requirements than XRC, NMR and cryo-EM. In this article, we describe best practices involved in the process of RNA and RNA-protein sample preparation, SAXS data collection, data analysis, and structural model building.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Escherichia coli/genética , Pliegue del ARN , ARN de Transferencia Aminoácido-Específico/química , Aminoacilación de ARN de Transferencia , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/genética , Cromatografía en Gel , Escherichia coli/metabolismo , Modelos Moleculares , Electroforesis en Gel de Poliacrilamida Nativa , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia Aminoácido-Específico/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Differential scanning fluorimetry (DSF) is a fluorescence-based assay to evaluate protein stability by determining protein melting temperatures. Here, we describe the application of DSF to investigate aminoacyl-tRNA synthetase (AARS) stability and interaction with ligands. Employing three bacterial AARS enzymes as model systems, methods are presented here for the use of DSF to measure the apparent temperatures at which AARSs undergo melting transitions, and the effect of AARS substrates and inhibitors. One important observation is that the extent of temperature stability realized by an AARS in response to a particular bound ligand cannot be predicted a priori. The DSF method thus serves as a rapid and highly quantitative approach to measure AARS stability, and the ability of ligands to influence the temperature at which unfolding transitions occur.