Isolation of translationally controlled mRNAs by differential screening - PubMed (original) (raw)
. 2000 Aug;14(11):1641-52.
doi: 10.1096/fj.14.11.1641.
Affiliations
- PMID: 10928999
- DOI: 10.1096/fj.14.11.1641
Isolation of translationally controlled mRNAs by differential screening
W Mikulits et al. FASEB J. 2000 Aug.
Abstract
Translationalregulation plays an important role in the control of gene expression. Changes in translation initiation rates are the most common translation-regulating mechanisms, resulting in alterations in mRNA loading of ribosomes. This differential mobilization of mRNAs onto polyribosomes was used in differential screening to directly identify cDNAs whose transcripts are translationally controlled during antigenic stimulation of primary human T lymphocytes. Ribosome-free and polysome-bound mRNAs were prepared from quiescent and activated T cells and used as templates to synthesize four cDNA pools. These in turn were used as probes to hybridize four identical replicas of a T cell library or, alternatively, four cDNA arrays. Translational activation was indicated by redistribution of the hybridization signals from the ribosome-free fraction in resting T cells to the polysome-associated fraction in activated T cells. Translational repression corresponded to the opposite hybridization pattern. Fifty-two cDNAs were identified as translationally controlled by screening 472 genes in a cDNA array; 12 additional ones were obtained by screening a cDNA library. Several of the transcripts corresponded to mRNAs previously reported to be translationally controlled, thus validating the method. For the majority, however, such regulation had not yet been described. Translational control was verified for representative examples by demonstrating the redistribution of the corresponding mRNAs on polysome gradients in response to T cell activation. Our strategy therefore provides an efficient tool to directly isolate or identify translationally controlled mRNAs in a variety of physiological situations. Moreover, differential screening using arrays enables simultaneous analysis of both transcriptional and translational regulation, further enhancing the power of gene expression analysis.
Similar articles
- Translational control: a general mechanism for gene regulation during T cell activation.
Garcia-Sanz JA, Mikulits W, Livingstone A, Lefkovits I, Müllner EW. Garcia-Sanz JA, et al. FASEB J. 1998 Mar;12(3):299-306. doi: 10.1096/fasebj.12.3.299. FASEB J. 1998. PMID: 9506473 - Messenger RNA translation state: the second dimension of high-throughput expression screening.
Zong Q, Schummer M, Hood L, Morris DR. Zong Q, et al. Proc Natl Acad Sci U S A. 1999 Sep 14;96(19):10632-6. doi: 10.1073/pnas.96.19.10632. Proc Natl Acad Sci U S A. 1999. PMID: 10485877 Free PMC article. - Global mRNA stabilization preferentially linked to translational repression during the endoplasmic reticulum stress response.
Kawai T, Fan J, Mazan-Mamczarz K, Gorospe M. Kawai T, et al. Mol Cell Biol. 2004 Aug;24(15):6773-87. doi: 10.1128/MCB.24.15.6773-6787.2004. Mol Cell Biol. 2004. PMID: 15254244 Free PMC article. - Isolation of plant polysomal mRNA by differential centrifugation and ribosome immunopurification methods.
Mustroph A, Juntawong P, Bailey-Serres J. Mustroph A, et al. Methods Mol Biol. 2009;553:109-26. doi: 10.1007/978-1-60327-563-7_6. Methods Mol Biol. 2009. PMID: 19588103 Review. - TOPs and their regulation.
Hamilton TL, Stoneley M, Spriggs KA, Bushell M. Hamilton TL, et al. Biochem Soc Trans. 2006 Feb;34(Pt 1):12-6. doi: 10.1042/BST20060012. Biochem Soc Trans. 2006. PMID: 16246169 Review.
Cited by
- Integrative temporal multi-omics reveals uncoupling of transcriptome and proteome during human T cell activation.
Weerakoon H, Mohamed A, Wong Y, Chen J, Senadheera B, Haigh O, Watkins TS, Kazakoff S, Mukhopadhyay P, Mulvenna J, Miles JJ, Hill MM, Lepletier A. Weerakoon H, et al. NPJ Syst Biol Appl. 2024 Feb 28;10(1):21. doi: 10.1038/s41540-024-00346-4. NPJ Syst Biol Appl. 2024. PMID: 38418561 Free PMC article. - mTOR-dependent translation drives tumor infiltrating CD8+ effector and CD4+ Treg cells expansion.
De Ponte Conti B, Miluzio A, Grassi F, Abrignani S, Biffo S, Ricciardi S. De Ponte Conti B, et al. Elife. 2021 Nov 17;10:e69015. doi: 10.7554/eLife.69015. Elife. 2021. PMID: 34787568 Free PMC article. - Polysome profiling followed by RNA-seq of cardiac differentiation stages in hESCs.
Pereira IT, Spangenberg L, Robert AW, Amorín R, Stimamiglio MA, Naya H, Dallagiovanna B. Pereira IT, et al. Sci Data. 2018 Dec 4;5:180287. doi: 10.1038/sdata.2018.287. Sci Data. 2018. PMID: 30512016 Free PMC article. - Insights into the mechanisms of eukaryotic translation gained with ribosome profiling.
Andreev DE, O'Connor PB, Loughran G, Dmitriev SE, Baranov PV, Shatsky IN. Andreev DE, et al. Nucleic Acids Res. 2017 Jan 25;45(2):513-526. doi: 10.1093/nar/gkw1190. Epub 2016 Dec 6. Nucleic Acids Res. 2017. PMID: 27923997 Free PMC article. Review. - Polysome Profiling Links Translational Control to the Radioresponse of Glioblastoma Stem-like Cells.
Wahba A, Rath BH, Bisht K, Camphausen K, Tofilon PJ. Wahba A, et al. Cancer Res. 2016 May 15;76(10):3078-87. doi: 10.1158/0008-5472.CAN-15-3050. Epub 2016 Mar 22. Cancer Res. 2016. PMID: 27005284 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources