Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo, Japan.
Genome Res. 2012 May;22(5):947-56. doi: 10.1101/gr.130559.111. Epub 2012 Feb 27.
Mammalian genomes produce huge numbers of noncoding RNAs (ncRNAs). However, the functions of most ncRNAs are unclear, and novel techniques that can distinguish functional ncRNAs are needed. Studies of mRNAs have revealed that the half-life of each mRNA is closely related to its physiological function, raising the possibility that the RNA stability of an ncRNA reflects its function. In this study, we first determined the half-lives of 11,052 mRNAs and 1418 ncRNAs in HeLa Tet-off (TO) cells by developing a novel genome-wide method, which we named 5'-bromo-uridine immunoprecipitation chase-deep sequencing analysis (BRIC-seq). This method involved pulse-labeling endogenous RNAs with 5'-bromo-uridine and measuring the ongoing decrease in RNA levels over time using multifaceted deep sequencing. By analyzing the relationship between RNA half-lives and functional categories, we found that RNAs with a long half-life (t(1/2) ≥ 4 h) contained a significant proportion of ncRNAs, as well as mRNAs involved in housekeeping functions, whereas RNAs with a short half-life (t(1/2) < 4 h) included known regulatory ncRNAs and regulatory mRNAs. The stabilities of a significant set of short-lived ncRNAs are regulated by external stimuli, such as retinoic acid treatment. In particular, we identified and characterized several novel long ncRNAs involved in cell proliferation from the group of short-lived ncRNAs. We designated this novel class of ncRNAs with a short half-life as Short-Lived noncoding Transcripts (SLiTs). We propose that the strategy of monitoring RNA half-life will provide a powerful tool for investigating hitherto functionally uncharacterized regulatory RNAs.
哺乳动物基因组产生大量的非编码 RNA（ncRNA）。然而，大多数 ncRNA 的功能尚不清楚，需要开发能够区分功能性 ncRNA 的新技术。对 mRNAs 的研究表明，每个 mRNA 的半衰期与其生理功能密切相关，这使得 ncRNA 的 RNA 稳定性反映其功能的可能性增加。在这项研究中，我们首先通过开发一种新的全基因组方法，即 5'-溴尿嘧啶免疫沉淀追踪深度测序分析（BRIC-seq），确定了 HeLa Tet-off（TO）细胞中 11052 个 mRNA 和 1418 个 ncRNA 的半衰期。该方法涉及用 5'-溴尿嘧啶脉冲标记内源性 RNA，并使用多方面的深度测序来测量 RNA 水平随时间的持续下降。通过分析 RNA 半衰期与功能类别之间的关系，我们发现半衰期长（t(1/2)≥4 h）的 RNA 中包含大量 ncRNA，以及参与维持细胞功能的 mRNA，而半衰期短（t(1/2)<4 h）的 RNA 中包含已知的调节性 ncRNA 和调节性 mRNA。一组短半衰期 ncRNA 的稳定性受到外部刺激的调节，如视黄酸处理。特别是，我们从短半衰期 ncRNA 中鉴定并表征了几个参与细胞增殖的新型长 ncRNA。我们将这一类半衰期短的 ncRNA 命名为短寿命非编码转录物（SLiTs）。我们提出，监测 RNA 半衰期的策略将为研究迄今为止功能未被表征的调节性 RNA 提供一种强大的工具。
