Targeted m(6)A Reader Proteins To Study Epitranscriptomic Regulation of Single RNAs.
Targeted m(6)A Reader Proteins To Study Epitranscriptomic Regulation of Single RNAs.
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DOI：
10.1021/jacs.8b05012
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发表时间：
2018-09-26
影响因子：
15
通讯作者：
Dickinson BC
中科院分区：
文献类型：
--
作者：
Rauch S;He C;Dickinson BC
RNA的转录后基因表达调控已经成为控制哺乳动物蛋白质生产的关键因素。RNA运输、翻译效率和稳定性都在转录水平上受到控制。例如，除了通常已知的加帽、剪接和聚腺苷酸化的加工步骤之外，RNA可以被化学修饰。在真核生物中，N6-甲基腺苷（m6 A）是最普遍的mRNA修饰。虽然m6 A的写入器、擦除器和读取器正在全细胞水平上迅速被发现和研究，但它们在调节甲基化RNA转录物方面的竞争性相互作用尚未阐明。为了解决这一限制，我们报告了可编程dPspCas 13 b-m6 A阅读器蛋白的开发，以研究特定阅读器对活细胞中单个转录本的调节作用。我们将两种最充分表征的m6 A阅读器蛋白YTHDF 1和YTHDF 2与无催化活性的PspCas 13 b蛋白融合，该蛋白可以使用向导RNA（gRNA）互补性将阅读器靶向特定的感兴趣RNA。然后，我们证明，融合的阅读器蛋白各自保留其报告的功能作用的报告构建：YTHDF 2诱导降解和YTHDF 1增强翻译。最后，我们表明该系统可以靶向细胞内的内源性mRNA转录本，使用YTHDF 2作为范例，我们发现与YTHDF 2的拴系导致靶转录本的衰减。开发基于dCas 13 b的工具来研究内源性RNA的调控，将极大地增强我们对RNA调控如何在单个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变或增强的蛋白质生产，将在合成生物学应用中找到实用性，旨在控制RNA水平的遗传信息流。
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, N6-methyladenosine (m 6A) is the most prevalent mRNA modification. While the writers, erasers, and readers for m6A 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-m6A reader proteins to investigate the regulatory effects of specific readers on single transcripts in live cells. We fused the two most well-characterized m6A 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.
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影响因子：
30.3
作者：
Kennedy EM;Bogerd HP;Kornepati AV;Kang D;Ghoshal D;Marshall JB;Poling BC;Tsai K;Gokhale NS;Horner SM;Cullen BR
通讯作者：
Cullen BR
YTHDF2 通过直接招募 CCR4-NOT 去腺苷酶复合物来破坏含有 m(6)A 的 RNA 的稳定性。
影响因子：
16.6
作者：
Du, Hao;Zhao, Ya;He, Jinqiu;Zhang, Yao;Xi, Hairui;Liu, Mofang;Ma, Jinbiao;Wu, Ligang
通讯作者：
Wu, Ligang
影响因子：
48
作者：
Linder, Bastian;Grozhik, Anya V.;Olarerin-George, Anthony O.;Meydan, Cem;Mason, Christopher E.;Jaffrey, Samie R.
通讯作者：
Jaffrey, Samie R.
DOI：
10.1126/science.1260793
发表时间：
2015-02-06
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Battle A;Khan Z;Wang SH;Mitrano A;Ford MJ;Pritchard JK;Gilad Y
通讯作者：
Gilad Y
DOI：
10.1126/science.aaf5573
发表时间：
2016-08-05
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Abudayyeh OO;Gootenberg JS;Konermann S;Joung J;Slaymaker IM;Cox DB;Shmakov S;Makarova KS;Semenova E;Minakhin L;Severinov K;Regev A;Lander ES;Koonin EV;Zhang F
通讯作者：
Zhang F