Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals

Whole transcriptome analyses using tiling microarrays (Bertone et al. 2004) and deep sequencing (Nagalakshmi et al. 2008) have revealed huge numbers of novel transcripts, including long and short noncoding RNAs (ncRNAs). The ratio of noncoding to protein-coding genomic regions increases as a function of developmental complexity (Mattick 2004), suggesting that revealing the functions of ncRNAs transcribed from noncoding genomic regions is important for understanding genome function in higher organisms. The ncRNAs can be roughly classified into two groups: small transcripts, such as microRNAs and piwi-interacting RNAs (piRNAs), and long transcripts (Prasanth and Spector 2007). Although the biological importance of small ncRNAs has been documented in recent years, the physiological functions of long ncRNAs (lncRNAs) are poorly understood. Recently, significant efforts have been applied to reveal the function of lncRNAs. Several approaches have succeeded in identifying dozens of functional lncRNAs (Guttman et al. 2009). However, the biological functions of the vast majority of lncRNAs remain unclear. Thus, novel properties that can distinguish functional ncRNAs from transcriptional noise are required. Numerous studies of mRNAs have revealed that changing the abundance of transcripts by regulated RNA degradation is a critical step in the control of various biological pathways (Keene 2010). It has been estimated that the mRNA abundance of 5%–10% of human genes is controlled through the regulation of RNA stability (Bolognani and Perrone-Bizzozero 2008). It has been proposed that the specific half-life of each mRNA is closely related to its physiological function (Lam et al. 2001; Yang et al. 2003; Raghavan and Bohjanen 2004; Sharova et al. 2009; Rabani et al. 2011; Schwanhausser et al. 2011). Although mRNAs of most housekeeping genes have long half-lives, mRNAs of many regulatory genes, which encode proteins that are required for only a limited time in the cell—such as cell cycle regulators, factors responsible for responses to external stimuli, and regulators of growth or differentiation—often have short half-lives. Moreover, most transcriptionally inducible genes are disproportionately classified into the group of genes with rapid mRNA turnover. It is possible, therefore, that the RNA stability of noncoding transcripts also reflects their functions. Traditionally, RNA decay has been assessed by blocking global transcription with transcriptional inhibitors, e.g., actinomycin D (ActD), and subsequently monitoring ongoing RNA decay over time. However, inhibitor-mediated global transcriptional arrest has a profoundly disruptive impact on cellular physiology and interferes with the precise determination of the RNA degradation rate (Blattner et al. 2000; Friedel et al. 2009). Here, we present a novel inhibitor-free method (5′-bromo-uridine immunoprecipitation chase, BRIC) that enables measurement of RNA decay under nondisruptive conditions. Determination of the half-lives of whole transcripts by BRIC, combined with multifaceted deep sequencing (BRIC-seq), suggest that there is a relationship between the stability of ncRNAs, as well as mRNAs, and their physiological functions.