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Determination of tRNA aminoacylation levels by high-throughput sequencing
Transfer RNA (tRNA) decodes mRNA codons when aminoacylated (charged) with an amino acid at its 3′ end. Charged tRNAs turn over rapidly in cells, and variations in charged tRNA fractions are known to be a useful parameter in cellular responses to stress. tRNA charging fractions can be measured for in...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737633/ https://www.ncbi.nlm.nih.gov/pubmed/28586482 http://dx.doi.org/10.1093/nar/gkx514 |
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author | Evans, Molly E. Clark, Wesley C. Zheng, Guanqun Pan, Tao |
author_facet | Evans, Molly E. Clark, Wesley C. Zheng, Guanqun Pan, Tao |
author_sort | Evans, Molly E. |
collection | PubMed |
description | Transfer RNA (tRNA) decodes mRNA codons when aminoacylated (charged) with an amino acid at its 3′ end. Charged tRNAs turn over rapidly in cells, and variations in charged tRNA fractions are known to be a useful parameter in cellular responses to stress. tRNA charging fractions can be measured for individual tRNA species using acid denaturing gels, or comparatively at the genome level using microarrays. These hybridization-based approaches cannot be used for high resolution analysis of mammalian tRNAs due to their large sequence diversity. Here we develop a high-throughput sequencing method that enables accurate determination of charged tRNA fractions at single-base resolution (Charged DM-tRNA-seq). Our method takes advantage of the recently developed DM-tRNA-seq method, but includes additional chemical steps that specifically remove the 3′A residue in uncharged tRNA. Charging fraction is obtained by counting the fraction of A-ending reads versus A+C-ending reads for each tRNA species in the same sequencing reaction. In HEK293T cells, most cytosolic tRNAs are charged at >80% levels, whereas tRNA(Ser) and tRNA(Thr) are charged at lower levels. These low charging levels were validated using acid denaturing gels. Our method should be widely applicable for investigations of tRNA charging as a parameter in biological regulation. |
format | Online Article Text |
id | pubmed-5737633 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-57376332018-01-04 Determination of tRNA aminoacylation levels by high-throughput sequencing Evans, Molly E. Clark, Wesley C. Zheng, Guanqun Pan, Tao Nucleic Acids Res Methods Online Transfer RNA (tRNA) decodes mRNA codons when aminoacylated (charged) with an amino acid at its 3′ end. Charged tRNAs turn over rapidly in cells, and variations in charged tRNA fractions are known to be a useful parameter in cellular responses to stress. tRNA charging fractions can be measured for individual tRNA species using acid denaturing gels, or comparatively at the genome level using microarrays. These hybridization-based approaches cannot be used for high resolution analysis of mammalian tRNAs due to their large sequence diversity. Here we develop a high-throughput sequencing method that enables accurate determination of charged tRNA fractions at single-base resolution (Charged DM-tRNA-seq). Our method takes advantage of the recently developed DM-tRNA-seq method, but includes additional chemical steps that specifically remove the 3′A residue in uncharged tRNA. Charging fraction is obtained by counting the fraction of A-ending reads versus A+C-ending reads for each tRNA species in the same sequencing reaction. In HEK293T cells, most cytosolic tRNAs are charged at >80% levels, whereas tRNA(Ser) and tRNA(Thr) are charged at lower levels. These low charging levels were validated using acid denaturing gels. Our method should be widely applicable for investigations of tRNA charging as a parameter in biological regulation. Oxford University Press 2017-08-21 2017-06-06 /pmc/articles/PMC5737633/ /pubmed/28586482 http://dx.doi.org/10.1093/nar/gkx514 Text en © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Evans, Molly E. Clark, Wesley C. Zheng, Guanqun Pan, Tao Determination of tRNA aminoacylation levels by high-throughput sequencing |
title | Determination of tRNA aminoacylation levels by high-throughput sequencing |
title_full | Determination of tRNA aminoacylation levels by high-throughput sequencing |
title_fullStr | Determination of tRNA aminoacylation levels by high-throughput sequencing |
title_full_unstemmed | Determination of tRNA aminoacylation levels by high-throughput sequencing |
title_short | Determination of tRNA aminoacylation levels by high-throughput sequencing |
title_sort | determination of trna aminoacylation levels by high-throughput sequencing |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737633/ https://www.ncbi.nlm.nih.gov/pubmed/28586482 http://dx.doi.org/10.1093/nar/gkx514 |
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