title: Methodology for the High-Throughput Identification and Characterization of tRNA Variants That Are Substrates for a tRNA Decay Pathway.
abstract: The rapid tRNA decay (RTD) pathway is a tRNA quality control pathway known to degrade several specific hypomodified or destabilized tRNAs in the yeast Saccharomyces cerevisiae. In this chapter, we describe seven methods for identifying RTD substrates, with a focus on two new approaches: a high-throughput approach that utilizes a suppressor tRNA library, fluorescence-activated cell sorting, and deep sequencing, and has greatly expanded the known range of RTD substrates; and a poison primer extension assay that allows for the measurement of levels of suppressor tRNA variants, even in the presence of highly similar endogenous tRNAs. We also discuss different applications of the use of the high-throughput and poison primer extension methodologies for different problems in tRNA biology.
authors.affiliation: Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, New York, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Payea
authors.affiliation: Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, New York, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Guy
authors.affiliation: Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, New York, USA. Electronic address: eric_phizicky@urmc.rochester.edu.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Phizicky
mesh_terms: D005434:Flow Cytometry / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D020871:RNA Stability / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae
chemical_list: D012343:RNA, Transfer
keywords: High throughput; Poison primer extension; RNA-ID; Rapid tRNA decay; SUP4(oc); Yeast