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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...

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Autores principales: Evans, Molly E., Clark, Wesley C., Zheng, Guanqun, Pan, Tao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2017
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.
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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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