J Am Chem Soc. 2018 Sep 26;140(38):11974-11981. doi: 10.1021/jacs.8b05012. Epub 2018 Sep 18.
Post-transcriptional gene expression regulation of RNA has emerged as a key factor that controls mammalian protein production. RNA trafficking, translation efficiency, and stability are all controlled at the transcript level. For example, in addition to the commonly known processing steps of capping, splicing, and polyadenylation, RNA can be chemically modified. In eukaryotes, N-methyladenosine (mA) is the most prevalent mRNA modification. While the writers, erasers, and readers for mA are rapidly being uncovered and studied at the whole-cell level, their competitive interplay to regulate methylated RNA transcripts has yet to be elucidated. To address this limitation, we report the development of programmable dPspCas13b-mA reader proteins to investigate the regulatory effects of specific readers on single transcripts in live cells. We fused the two most well-characterized mA reader proteins, YTHDF1 and YTHDF2, to a catalytically inactive PspCas13b protein, which can target the reader to a specific RNA of interest using guide RNA (gRNA) complementarity. We then demonstrate that the fused reader proteins each retain their reported functional role on a reporter construct: YTHDF2 induces degradation and YTHDF1 enhances translation. Finally, we show that the system can target endogenous mRNA transcripts within cells, using YTHDF2 as an exemplar, where we found tethering with YTHDF2 leads to decay of the target transcript. The development of dCas13b-based tools to study the regulation of endogenous RNAs will dramatically enhance our understanding of how RNA regulation occurs at the single RNA level. Additionally, our new tools, which permit transcript-specific mediated decay or enhanced protein production, will find utility in synthetic biology applications aimed at controlling genetic information flow at the RNA level.
RNA 的转录后基因表达调控已成为控制哺乳动物蛋白质产生的关键因素。RNA 的运输、翻译效率和稳定性都在转录水平上受到控制。例如，除了常见的加帽、剪接和多聚腺苷酸化等加工步骤外，RNA 还可以进行化学修饰。在真核生物中，N6-甲基腺苷（m6A）是最普遍的 mRNA 修饰。虽然 mA 的写入器、擦除器和读取器在全细胞水平上被迅速揭示和研究，但它们在调节甲基化 RNA 转录本方面的竞争相互作用尚未阐明。为了解决这一限制，我们报告了可编程 dPspCas13b-mA 读取器蛋白的开发，以研究特定读取器对活细胞中单转录本的调节作用。我们将两个研究最充分的 mA 读取器蛋白，YTHDF1 和 YTHDF2，融合到一个无催化活性的 PspCas13b 蛋白上，该蛋白可以使用向导 RNA（gRNA）互补性将读取器靶向特定的感兴趣 RNA。然后，我们证明融合的读取器蛋白各自保留了它们在报告构建体上的报告功能：YTHDF2 诱导降解，而 YTHDF1 增强翻译。最后，我们展示了该系统可以在细胞内靶向内源性 mRNA 转录本，以 YTHDF2 为例，我们发现与 YTHDF2 连接导致靶转录本的衰减。基于 dCas13b 的工具的开发将极大地增强我们对 RNA 调节如何在单个 RNA 水平上发生的理解。此外，我们的新工具允许转录特异性介导的衰减或增强的蛋白质生产，将在旨在控制 RNA 水平遗传信息流动的合成生物学应用中找到用途。
