Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA.
Chemical Biology Institute, Yale University, West Haven, CT 06516, USA.
Nucleic Acids Res. 2018 Aug 21;46(14):6996-7005. doi: 10.1093/nar/gky556.
Here, we describe an approach to enrich newly transcribed RNAs from primary mouse neurons using 4-thiouridine (s4U) metabolic labeling and solid phase chemistry. This one-step enrichment procedure captures s4U-RNA by using highly efficient methane thiosulfonate (MTS) chemistry in an immobilized format. Like solution-based methods, this solid-phase enrichment can distinguish mature RNAs (mRNA) with differential stability, and can be used to reveal transient RNAs such as enhancer RNAs (eRNAs) and primary microRNAs (pri-miRNAs) from short metabolic labeling. Most importantly, the efficiency of this solid-phase chemistry made possible the first large scale measurements of RNA polymerase II (RNAPII) elongation rates in mouse cortical neurons. Thus, our approach provides the means to study regulation of RNA metabolism in specific tissue contexts as a means to better understand gene expression in vivo.
在这里,我们描述了一种从原代小鼠神经元中富集新转录 RNA 的方法,该方法使用 4-硫代尿嘧啶 (s4U) 代谢标记和固相化学。这种一步富集程序通过在固定化形式中使用高效的甲烷硫代磺酸酯 (MTS) 化学来捕获 s4U-RNA。与基于溶液的方法一样,这种固相富集可以区分具有不同稳定性的成熟 RNA(mRNA),并且可用于从短期代谢标记中揭示瞬时 RNA,如增强子 RNA (eRNA) 和初级 microRNA (pri-miRNA)。最重要的是,这种固相化学的效率使得首次能够在小鼠皮质神经元中大规模测量 RNA 聚合酶 II (RNAPII) 延伸率。因此,我们的方法为在特定组织背景下研究 RNA 代谢的调控提供了手段,以便更好地理解体内基因表达。
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