Sequence-specific targeting of RNA.
Sequence-specific targeting of RNA.
复制标题
RNA 的序列特异性靶向。
DOI：
10.1016/j.ymeth.2022.06.007
复制
发表时间：
2022
期刊：
影响因子：
4.8
通讯作者：
A. Rentmeister
中科院分区：
文献类型：
--
作者：
Lukas Rösner;Dennis Reichert;Kristina Rau;N. Muthmann;A. Rentmeister
转录后修饰在真核细胞的翻译、剪接和 RNA 降解等多个过程中发挥着重要作用。为了研究特定修饰的功能，开发用于序列特异性 RNA 靶向的工具非常有意义。这项工作重点关注真核 mRNA 的两种丰富修饰，即 5'-帽鸟嘌呤-N7 位置的甲基化和内部 N6-甲基腺苷 (m6A)。我们描述了通过 RNA 结合蛋白对模型 RNA 转录本进行序列特异性靶向，例如核酸酶缺陷型 RNA 靶向 Cas9 (RCas9) 和与两种不同效应酶（双加氧酶 FTO 和鸟嘌呤 N7 甲基转移酶 Ecm1）融合的 Pumilio 同源结构域 (PumHD)。使用这个工具，我们能够以高特异性在各自的位置安装和去除甲基化。
Post-transcriptional modifications play an important role in several processes, including translation, splicing, and RNA degradation in eukaryotic cells. To investigate the function of specific modifications it is of high interest to develop tools for sequence-specific RNA-targeting. This work focuses on two abundant modifications of eukaryotic mRNA, namely methylation of the guanine-N7 position of the 5′-cap and internalN6-methyladenosine (m6A). We describe the sequence-specific targeting of model RNA transcripts via RNA-binding proteins, such as nuclease-deficient RNA-targeting Cas9 (RCas9) and the Pumilio homology domain (PumHD) fused to two different effector enzymes, the dioxygenase FTO and the guanine-N7 methyltransferase Ecm1. With this tool, we were able to install and remove the methylation at the respective positions with high specificity.