scholarly journals Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides

2019 ◽  
Author(s):  
Jamie R Wangen ◽  
Rachel Green
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Translation Termination ◽  
Premature Termination Codon ◽  
Ribosome Profiling ◽  
Termination Codon ◽  
Stop Codons ◽  
Genome Wide ◽  
A Genome

AbstractStop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418 treatment globally alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

scholarly journals Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jamie R Wangen ◽  
Rachel Green
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Translation Termination ◽  
Premature Termination Codon ◽  
Ribosome Profiling ◽  
Termination Codon ◽  
Stop Codons ◽  
Genome Wide ◽  
A Genome

Stop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418-induced miscoding alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

scholarly journals Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Kotchaphorn Mangkalaphiban ◽  
Feng He ◽  
Robin Ganesan ◽  
Chan Wu ◽  
Richard Baker ◽  
...  
Keyword(s):  
Stop Codon ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Genome Wide ◽  
A Genome ◽  
Stop Codon Readthrough

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3′-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5′ of the stop codon, six nucleotides 3′ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3′-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3′-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 and mRNA secondary structure in the 3′-UTR had milder effects. Additionally, we found low readthrough genes to have shorter 3′-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

scholarly journals Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

PLoS Genetics ◽  
2021 ◽  
Vol 17 (4) ◽  
pp. e1009538
Author(s):  
Kotchaphorn Mangkalaphiban ◽  
Feng He ◽  
Robin Ganesan ◽  
Chan Wu ◽  
Richard Baker ◽  
...  
Keyword(s):  
Stop Codon ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Genome Wide ◽  
A Genome ◽  
Stop Codon Readthrough

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

scholarly journals Distinct Paths To Stop Codon Reassignment by the Variant-Code Organisms Tetrahymena and Euplotes

2006 ◽  
Vol 26 (2) ◽  
pp. 438-447 ◽  
Author(s):  
Joe Salas-Marco ◽  
Hua Fan-Minogue ◽  
Adam K. Kallmeyer ◽  
Lawrence A. Klobutcher ◽  
Philip J. Farabaugh ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Yeast Cells ◽  
Universal Genetic Code ◽  
Stop Codons ◽  
Codon Recognition ◽  
Ciliate Species ◽  
Stop Codon Recognition ◽  
Euplotes Octocarinatus

ABSTRACT The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed that changes in domain 1 of ciliate eRF1s are responsible for altered stop codon recognition, this has never been demonstrated in vivo. To carry out such an analysis, we made hybrid proteins that contained eRF1 domain 1 from either Tetrahymena thermophila or Euplotes octocarinatus fused to eRF1 domains 2 and 3 from Saccharomyces cerevisiae. We found that the Tetrahymena hybrid eRF1 efficiently terminated at all three stop codons when expressed in yeast cells, indicating that domain 1 is not the sole determinant of stop codon recognition in Tetrahymena species. In contrast, the Euplotes hybrid facilitated efficient translation termination at UAA and UAG codons but not at the UGA codon. Together, these results indicate that while domain 1 facilitates stop codon recognition, other factors can influence this process. Our findings also indicate that these two ciliate species used distinct approaches to diverge from the universal genetic code.

scholarly journals Ribosome recycling factor ABCE1 depletion inhibits nonsense-mediated mRNA decay by promoting stop codon readthrough

2019 ◽  
Author(s):  
Giuditta Annibaldis ◽  
René Dreos ◽  
Michal Domanski ◽  
Sarah Carl ◽  
Oliver Mühlemann
Keyword(s):  
Mrna Decay ◽  
Stop Codon ◽  
Translation Termination ◽  
Ribosome Profiling ◽  
List Type ◽  
Ribosome Recycling Factor ◽  
Stop Codons ◽  
Nonsense Mediated Mrna Decay ◽  
Ribosome Disassembly ◽  
Stop Codon Readthrough

SUMMARYNonsense-mediated mRNA decay (NMD) is an essential post-transcriptional surveillance pathway in vertebrates that appears to be mechanistically linked with translation termination. To gain more insight into this connection, we interfered with translation termination by depleting human cells of the ribosome recycling factor ABCE1, which resulted in an upregulation of many but not all endogenous NMD-sensitive mRNAs. Notably, the suppression of NMD on these mRNAs occurs at a step prior to their SMG6-mediated endonucleolytic cleavage. Ribosome profiling revealed that ABCE1 depletion results in ribosome stalling at stop codons and increased ribosome occupancy in 3’ UTRs, indicative of enhanced stop codon readthrough or re-initiation. Using reporter genes, we further demonstrate that the absence of ABCE1 indeed increases the rate of readthrough, which would explain the observed NMD inhibition, since enhanced readthrough has been previously shown to render NMD-sensitive transcripts resistant to NMD by displacing NMD triggering factors like UPF1 and exon junction complexes (EJCs) from the 3’ UTR. Collectively, our results show that improper ribosome disassembly interferes with proper NMD activation.HighlightsABCE1 knockdown suppresses NMD of many NMD-sensitive mRNAsThe observed NMD inhibition occurs at a stage prior to SMG6-mediated cleavage of the mRNAABCE1 depletion enhances ribosome occupancy at stop codons and in the 3’ UTRABCE1 depletion enhances readthrough of the stop codonEnhanced readthrough inhibits NMD, presumably by clearing the 3’ UTR of NMD factors

scholarly journals Engineered transfer RNAs for suppression of premature termination codons

2018 ◽  
Author(s):  
John D. Lueck ◽  
Jae Seok Yoon ◽  
Alfredo Perales-Puchalt ◽  
Adam L. Mackey ◽  
Daniel T. Infield ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Premature Termination ◽  
Translation Termination ◽  
Genetic Disorders ◽  
Ribosome Profiling ◽  
Inherited Disease ◽  
Transfer Rnas ◽  
Premature Termination Codons ◽  
Nonsense Codons

ABSTRACTPremature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. We present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identified ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within the cystic fibrosis transmembrane conductance regulator (CFTR).

scholarly journals Genetic screens identify connections between ribosome recycling and nonsense mediated decay

2021 ◽  
Author(s):  
Karole N D'Orazio ◽  
Laura N. Lessen ◽  
Anthony J. Veltri ◽  
Zachary Neiman ◽  
Miguel E. Pacheco ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Premature Termination ◽  
Stop Codon ◽  
Premature Termination Codon ◽  
Termination Codon ◽  
Genetic Screens ◽  
Regulatory Pathway ◽  
Nonsense Mediated Decay ◽  
Stop Codons ◽  
Reported Data

The decay of messenger RNA with a premature termination codon (PTC) by nonsense mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway in mammals. NMD is typically triggered by the ribosome terminating at a stop codon that is aberrantly distant from the poly-A tail. Here, we use a fluorescence screen to identify factors involved in NMD in S. cerevisiae . In addition to the known NMD factors, including the entire UPF family (UPF1, UPF2 and UPF3), as well as NMD4 and EBS1 , we identify factors known to function in post-termination recycling and characterize their contribution to NMD. We then use a series of modified reporter constructs that block both elongating and scanning ribosomes downstream of stop codons and demonstrate that a deficiency in recycling of 80S ribosomes or 40S subunits stabilizes NMD substrates. These observations in S. cerevisiae expand on recently reported data in mammals indicating that the 60S recycling factor ABCE1 is important for NMD (1,2) by showing that increased activities of both elongating and scanning ribosomes (80S or 40S) in the 3’UTR correlate with a loss of NMD.

scholarly journals High-throughput 5’P sequencing enables the study of degradation-associated ribosome stalls

2020 ◽  
Author(s):  
Yujie Zhang ◽  
Vicent Pelechano
Keyword(s):  
High Throughput ◽  
Stop Codon ◽  
Translation Termination ◽  
Rna Degradation ◽  
Mrna Degradation ◽  
Ribosome Profiling ◽  
Sequencing Method ◽  
Rrna Depletion ◽  
Hydrophobic Amino Acids

ABSTRACTRNA degradation is critical for gene expression and mRNA quality control. mRNA degradation is connected to the translation process up to the degree that 5’-3’ mRNA degradation follows the las translating ribosome. Here we present an improved high-throughput 5’P degradome RNA sequencing method (HT-5Pseq). HT-5Pseq is easy, scalable and uses affordable duplex-specific nuclease based rRNA depletion. We investigate in vivo ribosome stalls focusing on translation termination. By comparing ribosome stalls identified by ribosome profiling, disome-seq and HT-5PSeq we identify that degradation-associated ribosome stalls are often enriched in Arg preceding the stop codon. On the contrary, mRNAs depleted for those stalls use more frequently TAA stop codon preceded by hydrophobic amino acids. Finally, we shown that termination stalls identified by HT-5Pseq, and not by other approaches, are associated to decreased mRNA stability. Our work suggests that ribosome stalls associated to mRNA decay can be easily captured by investigating the 5’P degradome.

Generation and Analysis of Target Genes Libraries of the Erythropoietic Transcription Factor GATA-1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1743-1743
Author(s):  
Eleni Z. Katsantoni ◽  
Sebastiaan Horsman ◽  
Michael J. Moorhouse ◽  
Victor C.L. de Jager ◽  
Peter van der Spek ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Target Genes ◽  
Genome Wide ◽  
Dna Libraries ◽  
A Genome ◽  
Search Tool ◽  
Or Gene ◽  
Novel Target

Abstract GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes. In erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes. In order to elucidate further the role of GATA-1 we applied an in vivo tagging methodology for the specific, quantitative biotinylation of this factor in mammalian cells (de Boer et al., 2003). We applied this method for identification of novel target genes of GATA-1 by performing pull-downs of crosslinked chromatin using streptavidin. We have also performed chromatin immunoprecipitations, where crosslinked chromatin was immunoprecipitated with antibodies against GATA-1, thus enriching for sequences bound in vivo by this factor in the immunoprecipitated DNA. Libraries of in vivo bound DNA targets were generated and a number of clones were sequenced. In order to facilitate the bioinformatics analysis of these libraries we generated TF Target Mapper (Transcription Factors Target Mapper). This is a BLAST search tool that allows rapid extraction of annotated information on genes around each hit and combines sequence cleaning/filtering, pattern searching and comparisons of the output list of genes or gene ontology IDs with user implemented lists. This tool was successfully applied to analyze sequences bound in vivo by the transcription factor GATA-1 and efficiently extracted information on genes around ChIPed sequences, thus identifying known and potentially novel GATA-1 gene targets. Using TF Target Mapper, 95 sequences were processed and annotated information on 372 genes 50kb upstream and downstream of each hit was extracted in 27 minutes. Among these genes, known targets of GATA-1, such as α-globin and ζ-globin, were readily identified by comparing to a list of known GATA-1 targets. This work is anticipated to provide a genome wide map of GATA-1 target genes in vivo. The identification of target genes and elucidation of their functions in hematopoiesis will allow the construction of complex transcriptional pathways that control lineage commitment and differentiation decisions.

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