Lenstra Tineke L, Coulon Antoine, Chow Carson C, Larson Daniel R
Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA.
Mol Cell. 2015 Nov 19;60(4):597-610. doi: 10.1016/j.molcel.2015.09.028. Epub 2015 Nov 5.
Eukaryotic transcription is pervasive, and many of the resulting RNAs are non-coding. It is unknown whether ubiquitous transcription is functional or simply reflects stochastic transcriptional noise. By single-molecule visualization of the dynamic interplay between coding and non-coding transcription at the GAL locus in living yeast cells, we show that antisense GAL10 ncRNA transcription can switch between functional and spurious under different conditions. During galactose induction, GAL10 sense transcription occurs in short stochastic bursts, which are unaffected by transcription of antisense GAL10 ncRNA, even when both are present simultaneously at the same locus. In contrast, when GAL10 is not induced, ncRNA transcription is critical to prevent transcriptional leakage of GAL1 and GAL10. Suppression of ncRNA transcription by strand-specific CRISPR/dCas9 results in transcriptional leakage of the inducer GAL1, leading to a more sensitive transcription activation threshold, an alteration of metabolic switching, and a fitness defect in competition experiments.
真核生物转录普遍存在，许多转录产生的RNA是非编码的。目前尚不清楚普遍存在的转录是功能性的还是仅仅反映了随机转录噪声。通过对活酵母细胞中GAL基因座上编码转录和非编码转录之间动态相互作用的单分子可视化，我们表明反义GAL10 ncRNA转录在不同条件下可以在功能性和假性之间切换。在半乳糖诱导期间，GAL10正义转录以短的随机爆发形式发生，即使两者同时存在于同一基因座，也不受反义GAL10 ncRNA转录的影响。相反，当GAL10未被诱导时，ncRNA转录对于防止GAL1和GAL10的转录泄漏至关重要。通过链特异性CRISPR/dCas9抑制ncRNA转录会导致诱导剂GAL1的转录泄漏，从而导致更敏感的转录激活阈值、代谢转换的改变以及竞争实验中的适应性缺陷。
