High resolution ribosome profiling reveals gene-specific details of UGA recoding in selenoprotein biosynthesis
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ABSTRACT:
PROVIDER: PRJNA607266 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11101354.fastq.gz | Fastqsanger.gz | |||
| SRR11101355.fastq.gz | Fastqsanger.gz | |||
| SRR11101356.fastq.gz | Fastqsanger.gz | |||
| SRR11101357.fastq.gz | Fastqsanger.gz | |||
| SRR11101360.fastq.gz | Fastqsanger.gz |
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Project description:SECIS binding protein 2 (SECISBP2) increases the efficiency of recoding of UGA codons as selenocysteine (Sec) during translation of mRNAs containing a selenocysteine insertion sequence (SECIS) in the 3’-untranslated region. Using ribosomal profiling, we have previously studied selenoprotein translation in hepatocyte-specific Secisbp2-deficient mouse liver and neuron-specific Secisbp2-mutant mouse brain. The use of organs carries the limitation of cellular heterogeneity with cells still expressing wild-type SECISBP2 that might potentially confound analyses and conclusions. To address this concern, we studied a haploid human HAP1 cell line carrying a non-functional SECISBP2 protein. These cells are still capable of metabolically incorporating 75Se into selenoproteins. We performed ribosomal profiling, and show that the efficiency of UGA recoding is gene-specific in SECISBP2- deficient cells. Analysis of ribosomes with UGA either at the A-site or the P-site revealed in a transcript-specific manner that SECISBP2 helps to recruit tRNASec and stabilizes mRNA. We propose a new measure to assess UGA/Sec read-through at codon resolution in all selenoproteins, i.e. the proportion of ribosomes carrying UGA in the P-site, pUGA. An additional, new observation is frame- shifting occurring after the UGA/Sec codon in GPX1, SELENOF, and SELENOW in HAP1 cells, a finding corroborated by reanalysis of neuron-specific Secisbp2-mutant brains.
2022-11-02 | GSE145465 | GEO
Project description:Co-translational incorporation of selenocysteine (Sec) into selenoproteins occurs at UGA codons in a process in which translational elongation competes with translational termination. Selenocysteine insertion sequence-binding protein 2 (SECISBP2) greatly enhances Sec incorporation into selenoproteins by interacting with the mRNA, ribosome, and elongation factor Sec (EFSEC). Ribosomal profiling allows to study the process of UGA re-coding in the physiological context of the cell and at the same time for all individual selenoproteins expressed in that cell. Using HAP1 cells expressing a mutant SECISBP2, we show here that high-resolution ribosomal profiling can be used to assess read-through efficiency at the UGA in all selenoproteins, including those with Sec close to the C-terminus. Analysis of ribosomes with UGA either at the A-site or the P-site revealed, in a transcript-specific manner, that SECISBP2 helps to recruit tRNASec and stabilize the mRNA. We propose to assess the effect of any perturbation of UGA read-through by determining the proportion of ribosomes carrying UGA in the P-site, pUGA. An additional, new observation is frameshifting that occurred 3' of the UGA/Sec codon in SELENOF and SELENOW in SECISBP2-mutant HAP1 cells, a finding corroborated by reanalysis of neuron-specific Secisbp2R543Q-mutant brains.
| S-EPMC9599142 | biostudies-literature
Project description:Mice carrying genomic deletions of SECIS1 or SECIS2 in the Selenop gene were euthanized and liver tissue was harvested and processed for RNA-Seq and ribosome profiling. From these studies, we determined the affect of SECIS1 and SECIS2 on UGA redifinition across the Selenop gene.
2017-08-22 | GSE102890 | GEO
Project description:Selenoproteins are a unique class of proteins that contain the 21st amino acid, selenocysteine (Sec). Addition of Sec into a protein is achieved by recoding of the UGA stop codon. All 25 mammalian selenoprotein mRNAs possess a 3' UTR stem-loop structure, the Selenocysteine Insertion Sequence (SECIS), which is required for Sec incorporation. It is widely believed that the SECIS is the major RNA element that controls Sec insertion, however recent findings in our lab suggest otherwise for Selenoprotein S (SelS). Here we report that the first 91 nucleotides of the SelS 3' UTR contain a proximal stem loop (PSL) and a conserved sequence we have named the SelS Positive UGA Recoding (SPUR) element. We developed a SelS-V5/UGA surrogate assay for UGA recoding, which was validated by mass spectrometry to be an accurate measure of Sec incorporation in cells. Using this assay, we show that point mutations in the SPUR element greatly reduce recoding in the reporter; thus, the SPUR is required for readthrough of the UGA-Sec codon. In contrast, deletion of the PSL increased Sec incorporation. This effect was reversed when the PSL was replaced with other stem-loops or an unstructured sequence, suggesting that the PSL does not play an active role in Sec insertion. Additional studies revealed that the position of the SPUR relative to the UGA-Sec codon is important for optimal UGA recoding. Our identification of the SPUR element in the SelS 3' UTR reveals a more complex regulation of Sec incorporation than previously realized.
| S-EPMC6844570 | biostudies-literature
Project description:We describe the effects of knocking out the Secisbp2 and Trsp genes on selenoprotein expression and translation in mouse liver
2016-10-30 | GSE84112 | GEO
Project description:Expression of selenoproteins requires the co-translational incorporation of selenocysteine (Sec) in response to an in-frame UGA codon. The machinery of UGA/Sec re-coding is complex and many factors affect the hierarchy of expression among selenoproteins, including modification of tRNA[Ser]Sec. Its hyper-modification in the anticodon stem loop is influenced by selenium bioavailability, and a mutation in adenosine 37 (A37) that abrogates isopentenylation, has a profound effect on selenoprotein expression in mice. Patients with mutations in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder and hence we wondered whether mutations in TRIT1 negatively affected the expression of selenoproteins. Fibroblasts from a patient carrying a pathogenic R323Q mutation in TRIT1 in homozygosity did not show decreased selenoprotein expression, although recombinant TRIT1R323Q had significantly reduced activity in vitro towards anticodon stem-loop substrates. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Selenoprotein expression as assessed by western blotting, 75Se metabolic labeling, and ribosomal profiling was not decreased despite the general reduction of N6-isopentenyl-adenosine in tRNAs. We show that 5-methylcarboxymethylation and 2’O-methylation of U34 occur independently of isopentenylation of A37 in tRNA[Ser]Sec. Reanalyzing previously published ribosomal profiling datasets, we demonstrate that (i) failure of 5-carboxymethylation at U34 is associated with reduced expression of GPX1, but not GPX4, and that (ii) FTSJ1 is not the elusive U34-2’O-methyltransferase involved in the methylation of tRNA[Ser]Sec.
2021-11-17 | GSE183923 | GEO
Project description:Re-coding of UGA codons as selenocysteine (Sec) codons in selenoproteins depends on a selenocysteine insertion sequence (SECIS) in the 3’ UTR of mRNAs of eukaryotic selenoproteins. SECIS-binding protein 2 (SECISBP2) increases the efficiency of this process. Pathogenic mutations in SECISBP2 reduce selenoprotein expression and lead to phenotypes associated with the reduction of deiodinase activities and selenoprotein N expression in humans. Two functions have been ascribed to SECISBP2: binding of SECIS elements in selenoprotein mRNAs and facilitation of co-translational Sec insertion. To separately probe both functions, we established here two mouse models carrying two pathogenic missense mutations in Secisbp2 previously identified in patients. We found that the C696R substitution in the RNAbinding domain abrogates SECIS binding and does not support selenoprotein translation above the level of a complete Secisbp2 null mutation. The R543Q missense substitution located in the selenocysteine insertion domain resulted in residual activity and caused reduced selenoprotein translation, as demonstrated by ribosomal profiling to determine the impact on UGA re-coding in individual selenoproteins. We found, however, that the R543Q variant is thermally unstable in vitro and completely degraded in the mouse liver in vivo, while being partially functional in the brain. The moderate impairment of selenoprotein expression in neurons led to astrogliosis and transcriptional induction of genes associated with immune responses. We conclude that differential SECISBP2 protein stability in individual cell types may dictate clinical phenotypes to a much greater extent than molecular interactions involving a mutated amino acid in SECISBP2 .
2019-07-26 | GSE119681 | GEO
Project description:Mammalian mRNAs are generated by complex and coordinated biogenesis pathways and acquire 5'-end m7G caps that play fundamental roles in processing and translation. Here we show that several selenoprotein mRNAs are not recognized efficiently by translation initiation factor eIF4E because they bear a hypermethylated cap. This cap modification is acquired via a 5’end maturation pathway similar to that of the small nucle(ol)ar RNAs (sn- and snoRNAs). Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs. We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated. Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo.
2014-05-14 | GSE57625 | GEO
Project description:In order to systematically assess the frequency and origin of stop codon recoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon recoding may occur with a rate as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon recoding events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon recoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon recoding by mass spectrometry revealed that temperature regulates the expression of cryptic sequences generated by stop codon recoding in E. coli. Overall, our findings suggest that the environment influences the accuracy of protein production which increases protein heterogeneity when the organisms need to adapt to new conditions.
2024-03-28 | GSE226936 | GEO
Project description:Chlamydia psittaci UGA Genome sequencing and assembly
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