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Identification of the determinants of tRNA function and susceptibility to rapid tRNA decay by high-throughput in vivo analysis.

ABSTRACT: Sequence variation in tRNA genes influences the structure, modification, and stability of tRNA; affects translation fidelity; impacts the activity of numerous isodecoders in metazoans; and leads to human diseases. To comprehensively define the effects of sequence variation on tRNA function, we developed a high-throughput in vivo screen to quantify the activity of a model tRNA, the nonsense suppressor SUP4oc of Saccharomyces cerevisiae. Using a highly sensitive fluorescent reporter gene with an ochre mutation, fluorescence-activated cell sorting of a library of SUP4oc mutant yeast strains, and deep sequencing, we scored 25,491 variants. Unexpectedly, SUP4oc tolerates numerous sequence variations, accommodates slippage in tertiary and secondary interactions, and exhibits genetic interactions that suggest an alternative functional tRNA conformation. Furthermore, we used this methodology to define tRNA variants subject to rapid tRNA decay (RTD). Even though RTD normally degrades tRNAs with exposed 5' ends, mutations that sensitize SUP4oc to RTD were found to be located throughout the sequence, including the anti-codon stem. Thus, the integrity of the entire tRNA molecule is under surveillance by cellular quality control machinery. This approach to assess activity at high throughput is widely applicable to many problems in tRNA biology.

SUBMITTER: Guy MP

PROVIDER: S-EPMC4117946 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Identification of the determinants of tRNA function and susceptibility to rapid tRNA decay by high-throughput in vivo analysis.

Guy Michael P MP Young David L DL Payea Matthew J MJ Zhang Xiaoju X Kon Yoshiko Y Dean Kimberly M KM Grayhack Elizabeth J EJ Mathews David H DH Fields Stanley S Phizicky Eric M EM

Genes & development 20140801 15

Sequence variation in tRNA genes influences the structure, modification, and stability of tRNA; affects translation fidelity; impacts the activity of numerous isodecoders in metazoans; and leads to human diseases. To comprehensively define the effects of sequence variation on tRNA function, we developed a high-throughput in vivo screen to quantify the activity of a model tRNA, the nonsense suppressor SUP4oc of Saccharomyces cerevisiae. Using a highly sensitive fluorescent reporter gene with an o ...[more]

PMID: 25085423

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Identification of the determinants of tRNA function and susceptibility to rapid tRNA decay by high-throughput in vivo analysis.

Publications

Identification of the determinants of tRNA function and susceptibility to rapid tRNA decay by high-throughput in vivo analysis.

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