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Genes Dev. 2014 Aug 1;28(15):1721-32. doi: 10.1101/gad.245936.114.

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

Author information

1
Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, New York, USA 14642;
2
Department of Genome Sciences.
3
Department of Genome Sciences, Department of Medicine, Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA 98195.
4
Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, New York, USA 14642; eric_phizicky@urmc.rochester.edu fields@uw.edu.

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.

KEYWORDS:

RTD; SUP4oc; high-throughput analysis; tRNA decay; tRNA function

PMID:
25085423
PMCID:
PMC4117946
DOI:
10.1101/gad.245936.114
[Indexed for MEDLINE]
Free PMC Article
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