title: A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA: Application to Stress-Induced Reprogramming of tRNA Modifications.
abstract: Here we describe an analytical platform for systems-level quantitative analysis of modified ribonucleosides in any RNA species, with a focus on stress-induced reprogramming of tRNA as part of a system of translational control of cell stress response. This chapter emphasizes strategies and caveats for each of the seven steps of the platform workflow: (1) RNA isolation, (2) RNA purification, (3) RNA hydrolysis to individual ribonucleosides, (4) chromatographic resolution of ribonucleosides, (5) identification of the full set of modified ribonucleosides, (6) mass spectrometric quantification of ribonucleosides, (6) interrogation of ribonucleoside datasets, and (7) mapping the location of stress-sensitive modifications in individual tRNA molecules. We have focused on the critical determinants of analytical sensitivity, specificity, precision, and accuracy in an effort to ensure the most biologically meaningful data on mechanisms of translational control of cell stress response. The methods described here should find wide use in virtually any analysis involving RNA modifications.
authors.affiliation: Department of Microbiology, National University of Singapore, Singapore; Singapore-MIT Alliance for Research and Technology, Singapore.
authors.forename: Weiling Maggie
authors.initials: WM
authors.lastname: Cai
authors.affiliation: Department of Microbiology, National University of Singapore, Singapore; Singapore-MIT Alliance for Research and Technology, Singapore.
authors.forename: Yok Hian
authors.initials: YH
authors.lastname: Chionh
authors.affiliation: Singapore-MIT Alliance for Research and Technology, Singapore.
authors.forename: Fabian
authors.initials: F
authors.lastname: Hia
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Chen
authors.initials: C
authors.lastname: Gu
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Kellner
authors.affiliation: Singapore-MIT Alliance for Research and Technology, Singapore; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Megan E
authors.initials: ME
authors.lastname: McBee
authors.affiliation: Singapore-MIT Alliance for Research and Technology, Singapore; School of Biological Sciences, Nanyang Technological Institute, Singapore.
authors.forename: Chee Sheng
authors.initials: CS
authors.lastname: Ng
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Yan Ling Joy
authors.initials: YL
authors.lastname: Pang
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Erin G
authors.initials: EG
authors.lastname: Prestwich
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Kok Seong
authors.initials: KS
authors.lastname: Lim
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: I Ramesh
authors.initials: IR
authors.lastname: Babu
authors.affiliation: College of Nanoscale Engineering and Science, State University of New York, Albany, New York, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Begley
authors.affiliation: Singapore-MIT Alliance for Research and Technology, Singapore; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. Electronic address: pcdedon@mit.edu.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Dedon
mesh_terms: D013058:Mass Spectrometry / Q000379:methods*; D014176:Protein Biosynthesis / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics; D012263:Ribonucleosides / Q000737:chemistry* / Q000235:genetics
chemical_list: D012263:Ribonucleosides; D012343:RNA, Transfer
keywords: Mass spectrometry; Stress response; Systems biology; Translation; tRNA modifications