Abstract
The abundance of a transcript is determined by its rate of synthesis and its rate of degradation; however, global methods for quantifying RNA abundance cannot distinguish variation in these two processes. Here, we introduce RNA approach to equilibrium sequencing (RATE-seq), which uses in vivo metabolic labeling of RNA and approach to equilibrium kinetics, to determine absolute RNA degradation and synthesis rates. RATE-seq does not disturb cellular physiology, uses straightforward normalization with exogenous spike-ins, and can be readily adapted for studies in most organisms. We demonstrate the use of RATE-seq to estimate genome-wide kinetic parameters for coding and noncoding transcripts in Saccharomyces cerevisiae.
Highlights
Remodeling of gene expression is critical for a broad range of biological processes from the cell division cycle and embryo development (Schier 2007) to cellular responses to extracellular signals (Gasch et al 2000)
Genomic run on assays (García-Martínez et al 2004) provide a means of estimating mRNA synthesis rates; these methods require isolation of nuclei or permeabilization of cells, which likely compromises the physiology of cells
Approach to equilibrium labeling, using radiolabeling, was developed over 40 yr ago to estimate the rate of total mRNA turnover (Greenberg 1972) and was subsequently used to study individual transcripts using transcript-specific probes (Harpold et al 1981; Kim and Warner 1983)
Summary
Remodeling of gene expression is critical for a broad range of biological processes from the cell division cycle and embryo development (Schier 2007) to cellular responses to extracellular signals (Gasch et al 2000). A comprehensive understanding of the regulation of gene expression programs, and the development of mathematical models that explain the dynamics of gene expression, requires the accurate estimation of absolute rates of both RNA synthesis and RNA degradation in vivo. MRNA decay rates have been estimated using transcriptional inhibition (Wang et al 2002; Grigull et al 2004; Shalem et al 2008) using either temperature-sensitive alleles of RNA polymerase II or chemical inhibition of transcript production While these methods succeed in inhibiting transcript synthesis, they typically result in a stress response or cellular death (Nonet et al 1987) resulting in the estimation of mRNA decay rates that may have little physiological relevance. Genome-wide estimation of in vivo kinetic parameters using metabolic labeling of RNA with 4tU has been reported using different experimental designs. RATE-seq can be readily implemented in any organism, making it a generally applicable method for characterizing the steady-state in vivo kinetics of RNA with unprecedented resolution
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