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Translational control analysis by translationally active RNA capture/microarray analysis (TrIP–Chip)

We have developed a new approach to systematically study post-transcriptional regulation in a small number of cells. Actively translating mRNAs are associated with polysomes and the newly synthesized peptide chains are closely associated with molecular chaperones such as hsp70s, which assist in the...

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Detalles Bibliográficos
Autores principales: Kudo, Kenji, Xi, Yaguang, Wang, Yuan, Song, Bo, Chu, Edward, Ju, Jingyue, Russo, James J., Ju, Jingfang
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875024/
https://www.ncbi.nlm.nih.gov/pubmed/20123731
http://dx.doi.org/10.1093/nar/gkq024
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author Kudo, Kenji
Xi, Yaguang
Wang, Yuan
Song, Bo
Chu, Edward
Ju, Jingyue
Russo, James J.
Ju, Jingfang
author_facet Kudo, Kenji
Xi, Yaguang
Wang, Yuan
Song, Bo
Chu, Edward
Ju, Jingyue
Russo, James J.
Ju, Jingfang
author_sort Kudo, Kenji
collection PubMed
description We have developed a new approach to systematically study post-transcriptional regulation in a small number of cells. Actively translating mRNAs are associated with polysomes and the newly synthesized peptide chains are closely associated with molecular chaperones such as hsp70s, which assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones provide an anchor with which to separate actively translating mRNAs associated with polysomes from free mRNAs. Affinity capture beads were developed to capture hsp70 chaperones associated with the polysome complexes. The isolated actively translating mRNAs were used for high-throughput expression profiling analysis. Feasibility was demonstrated using an in vitro translation system with known translationally regulated mRNA transcript thymidylate synthase (TS). We further developed the approach using HCT-116 colon cancer cells with both TS and p53 as positive controls. The steady-state levels of TS and p53 mRNAs were unaltered after 5-fluorouracil treatment as assessed by real-time qRT-PCR analysis. In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased. These differences in translational rate were revealed with our new approach from 500 cells. This technology has the potential to make investigation of translational control feasible with limited quantities of clinical specimens.
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spelling pubmed-28750242010-05-24 Translational control analysis by translationally active RNA capture/microarray analysis (TrIP–Chip) Kudo, Kenji Xi, Yaguang Wang, Yuan Song, Bo Chu, Edward Ju, Jingyue Russo, James J. Ju, Jingfang Nucleic Acids Res Methods Online We have developed a new approach to systematically study post-transcriptional regulation in a small number of cells. Actively translating mRNAs are associated with polysomes and the newly synthesized peptide chains are closely associated with molecular chaperones such as hsp70s, which assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones provide an anchor with which to separate actively translating mRNAs associated with polysomes from free mRNAs. Affinity capture beads were developed to capture hsp70 chaperones associated with the polysome complexes. The isolated actively translating mRNAs were used for high-throughput expression profiling analysis. Feasibility was demonstrated using an in vitro translation system with known translationally regulated mRNA transcript thymidylate synthase (TS). We further developed the approach using HCT-116 colon cancer cells with both TS and p53 as positive controls. The steady-state levels of TS and p53 mRNAs were unaltered after 5-fluorouracil treatment as assessed by real-time qRT-PCR analysis. In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased. These differences in translational rate were revealed with our new approach from 500 cells. This technology has the potential to make investigation of translational control feasible with limited quantities of clinical specimens. Oxford University Press 2010-05 2010-01-31 /pmc/articles/PMC2875024/ /pubmed/20123731 http://dx.doi.org/10.1093/nar/gkq024 Text en © The Author(s) 2010. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Kudo, Kenji
Xi, Yaguang
Wang, Yuan
Song, Bo
Chu, Edward
Ju, Jingyue
Russo, James J.
Ju, Jingfang
Translational control analysis by translationally active RNA capture/microarray analysis (TrIP–Chip)
title Translational control analysis by translationally active RNA capture/microarray analysis (TrIP–Chip)
title_full Translational control analysis by translationally active RNA capture/microarray analysis (TrIP–Chip)
title_fullStr Translational control analysis by translationally active RNA capture/microarray analysis (TrIP–Chip)
title_full_unstemmed Translational control analysis by translationally active RNA capture/microarray analysis (TrIP–Chip)
title_short Translational control analysis by translationally active RNA capture/microarray analysis (TrIP–Chip)
title_sort translational control analysis by translationally active rna capture/microarray analysis (trip–chip)
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875024/
https://www.ncbi.nlm.nih.gov/pubmed/20123731
http://dx.doi.org/10.1093/nar/gkq024
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