Seryl-tRNA synthetase promotes translational readthrough via mRNA binding by involving the selenocysteine incorporation machinery
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ABSTRACT:
PROVIDER: PRJNA960646 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24267132.fastq.gz | Fastqsanger.gz | |||
| SRR24267133.fastq.gz | Fastqsanger.gz | |||
| SRR24267134.fastq.gz | Fastqsanger.gz | |||
| SRR24267135.fastq.gz | Fastqsanger.gz | |||
| SRR24267136.fastq.gz | Fastqsanger.gz |
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Project description:Translational readthrough of UGA stop codons by selenocysteine-specific tRNA (tRNASec) enables the synthesis of selenoproteins. Seryl-tRNA synthetase (SerRS) charges tRNASec with serine, which is modified into selenocysteine and delivered to the ribosome by a designated elongation factor (eEFSec in eukaryotes). Here we found that components of human selenocysteine incorporation machinery (SerRS, tRNASec, and eEFSec) also increased translational readthrough of non-selenocysteine genes, including VEGFA, to create C-terminally extended isoforms. SerRS recognizes target mRNAs through a stem-loop structure that resembles the variable loop of its cognate tRNAs. This function of SerRS depends on both its enzymatic activity and a vertebrate-specific domain. Through eCLIP-seq, we identified additional SerRS-interacting mRNAs as potential readthrough genes. Moreover, SerRS overexpression was sufficient to reverse premature termination caused by a pathogenic nonsense mutation. Our findings expand the repertoire of selenoprotein biosynthesis machinery and suggest an avenue for therapeutic targeting of nonsense mutations using endogenous factors.
2025-04-11 | GSE230349 | GEO
Project description:Translational readthrough of UGA stop codons by selenocysteine-specific tRNA (tRNASec) enables the synthesis of selenoproteins. Seryl-tRNA synthetase (SerRS) charges tRNASec with serine, which is modified into selenocysteine and delivered to the ribosome by a designated elongation factor (eEFSec in eukaryotes). Here we found that components of the human selenocysteine incorporation machinery (SerRS, tRNASec, and eEFSec) also increased translational readthrough of non-selenocysteine genes, including VEGFA, to create C-terminally extended isoforms. SerRS recognizes target mRNAs through a stem-loop structure that resembles the variable loop of its cognate tRNAs. This function of SerRS depends on both its enzymatic activity and a vertebrate-specific domain. Through eCLIP-seq, we identified additional SerRS-interacting mRNAs as potential readthrough genes. Moreover, SerRS overexpression was sufficient to reverse premature termination caused by a pathogenic nonsense mutation. Our findings expand the repertoire of selenoprotein biosynthesis machinery and suggest an avenue for therapeutic targeting of nonsense mutations using endogenous factors.
| S-EPMC10602924 | biostudies-literature
Project description:Selenium (Se) is an important trace element for many organisms and is incorporated into selenoproteins as selenocysteine (Sec). In eukaryotes, selenophosphate synthetase SPS2 is essential for Sec biosynthesis. In recent years, genetic disruptions of both Sec biosynthesis genes and selenoprotein genes have been investigated in different animal models, which provide important clues for understanding the Se metabolism and function in these organisms. However, a systematic study on the knockdown of SPS2 has not been performed in vivo. Herein, we conducted microarray experiments to study the transcriptome of fruit flies with knockdown of SPS2 in larval and adult stages. Several hundred differentially expressed genes were identified in each stage. Genes expression profiles of 12 fruit fly samples corresponding to larval and adult stages of SPS2 knockdown and control groups (3 replicates for each group) were analyzed using an Affymetrix Drosophila genome microarray.
2016-04-06 | E-GEOD-79924 | biostudies-arrayexpress
Project description:Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution. 12 samples of Drosophila ribosome profiling and poly(A)+ mRNA-seq, including technical replicates in S2 cells, and biological replicates of 0-2 hour embryos
2013-12-05 | E-GEOD-49197 | biostudies-arrayexpress
Project description:Selenophosphate, which is the active donor of selenium in selenocysteine (Sec) biosynthesis is synthesized from selenite and ATP by an enzyme designated as selenophosphate synthetase (SPS).There are two isoforms of SPS in higher eukaryotes,SPS1 and SPS2. Initially, both SPS1 and SPS2 were thought to be involved in selenophosphate synthesis. However, it was subsequently shown that only SPS2 catalyzes selenophosphate synthesis. Although SPS1 is an essential gene in Drosophila, its function has not been determined. To identify transcriptional profile of SPS1 knockdown cells, which could offer valuable clues to identify molecular mechanism of SPS1, we introduced RNAi using its cognate double-stranded RNA into Drosophila SL2 cells, and then microarray analysis was carried out. Experiment Overall Design: In order to find primary or secondary target gene which is regulated by SPS1,Drosophila SL2 cells were harvested at day1, day3 and day5 after SPS1 knockown for RNA extraction and hybridization on Affymetrix microarrays.
2009-08-22 | E-GEOD-17685 | biostudies-arrayexpress
Project description:Readthrough into the 3′ untranslated region (3′UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in C. elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes encounter nonoptimal codons, a feature of 3′UTR sequences. Dysfunction of GCN1 results in mRNA and proteome imbalance, increasingly perturbing transmembrane proteins and collagens during aging. These results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.
2023-06-09 | PXD037037 | Pride
Project description:Selenium (Se) is an important trace element for many organisms and is incorporated into selenoproteins as selenocysteine (Sec). In eukaryotes, selenophosphate synthetase SPS2 is essential for Sec biosynthesis. In recent years, genetic disruptions of both Sec biosynthesis genes and selenoprotein genes have been investigated in different animal models, which provide important clues for understanding the Se metabolism and function in these organisms. However, a systematic study on the knockdown of SPS2 has not been performed in vivo. Herein, we conducted microarray experiments to study the transcriptome of fruit flies with knockdown of SPS2 in larval and adult stages. Several hundred differentially expressed genes were identified in each stage.
2016-04-06 | GSE79924 | GEO
Project description:Readthrough into the 3′ untranslated region (3′UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in C. elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes encounter nonoptimal codons, a feature of 3′UTR sequences. Dysfunction of GCN1 results in mRNA and proteome imbalance, increasingly perturbing transmembrane proteins and collagens during aging. These results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.
2023-05-15 | GSE214396 | GEO
Project description:Selenophosphate, which is the active donor of selenium in selenocysteine (Sec) biosynthesis is synthesized from selenite and ATP by an enzyme designated as selenophosphate synthetase (SPS).There are two isoforms of SPS in higher eukaryotes,SPS1 and SPS2. Initially, both SPS1 and SPS2 were thought to be involved in selenophosphate synthesis. However, it was subsequently shown that only SPS2 catalyzes selenophosphate synthesis. Although SPS1 is an essential gene in Drosophila, its function has not been determined. To identify transcriptional profile of SPS1 knockdown cells, which could offer valuable clues to identify molecular mechanism of SPS1, we introduced RNAi using its cognate double-stranded RNA into Drosophila SL2 cells, and then microarray analysis was carried out.
2009-08-18 | GSE17685 | GEO
Project description:Transcription is a highly regulated process, and stress-induced changes in gene transcription have been shown to play a major role in responses and adaptation to stress. Numerous emerging genome-wide studies reveal prevalent transcription beyond known protein-coding gene loci, generating a variety of new classes of RNAs, most of unknown function. One such class, termed downstream of gene (DoG)-containing transcripts, was reported to result from transcriptional readthrough upon osmotic stress in human cell lines. However, how widespread the readthrough phenomenon is, and what its causes and consequences are, remain elusive. Here we present a systematic genome-wide mapping of transcriptional readthrough, using deep nuclear RNA-seq, comparing heat shock, osmotic and oxidative stress in NIH3T3 mouse fibroblast cells. We observe massive induction of transcriptional readthrough under all stress conditions, with significant, yet not complete overlap of readthrough-induced loci between different conditions. Importantly, our analyses suggest that stress-induced transcriptional readthrough is not a random failure process, but is rather differentially induced across different conditions. Additionally, analyzing public Pol-II occupancy data further supported our findings of stress-induced readthrough. We explore potential regulators and find a role for HSF1 in the induction of a subset of heat shock-induced readthrough transcripts. Furthermore, we examine genomic features of readthrough transcription, and observe a unique chromatin signature typical of DoG-producing regions, suggesting that readthrough transcription is associated with the maintenance of an open chromatin state.
2017-08-31 | GSE98906 | GEO