Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNA.
Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNA.
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具有优异特性的合成 mRNA，可模仿天然组蛋白 mRNA 的细胞内命运。
DOI：
10.1007/978-1-4939-3625-0_6
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Rhoads,RobertE
中科院分区：
文献类型：
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作者：
Su,Wei;Slevin,MichaelK;Marzluff,WilliamF;Rhoads,RobertE
由于DNA和组蛋白水平必须密切平衡细胞存活，组蛋白表达受到高度调节。复制依赖性组蛋白表达的调控主要在mRNA水平上实现，当DNA复制在s期被抑制时，mRNA被迅速移除。组蛋白mRNA降解开始于在末端尿苷转移酶（TUTase）催化的3 '茎环（SL）后添加多个尿苷（寡尿嘧啶化）。先前的研究表明，组蛋白mRNA的降解通过5 ‘→3 ’和3 ‘→5 ’两个过程发生，但由于缺乏既定的协议，很难分析其相对贡献。通过在5 ‘和3 ’端进行结构修饰，可以提高合成mRNA在培养细胞和全动物中的翻译效率和稳定性。在本章中，我们介绍了利用修饰的帽二核苷酸类似物来阻断含有规范组蛋白mRNA 3 ‘ SL的报告mRNA的5 ’→3 ‘降解和监测低聚尿嘧啶化和3 ’→5 '降解如何发生的方法。提出了包含组蛋白3 ‘ SL和修饰的帽类似物的报告mRNA的合成方案，监测mRNA的稳定性和从5 ’或3 '端单向降解，以及通过传统或深度测序从降解产物中检测寡核苷酸(U)束。
Since DNA and histone levels must be closely balanced for cell survival, histone expressions are highly regulated. The regulation of replication-dependent histone expression is mainly achieved at the mRNA level, as the mRNAs are rapidly removed when DNA replication is inhibited during S-phase. Histone mRNA degradation initiates with addition of multiple uridines (oligouridylation) following the 3′ stem-loop (SL) catalyzed by terminal uridyltransferase (TUTase). Previous studies showed that histone mRNA degradation occurs through both 5′ → 3′ and 3′ → 5′ processes, but the relative contributions are difficult to dissect due to lack of established protocols. The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by structural modifications at the both 5′ and 3′ termini. In this chapter, we present methods of utilizing modified cap dinucleotide analogs to block 5′ → 3′ degradation of a reporter mRNA containing canonical histone mRNA 3′ SL and monitoring how oligouridylation and 3′ → 5′ degradation occur. Protocols are presented for synthesis of reporter mRNA containing the histone 3′ SL and modified cap analogs, monitoring mRNA stability and unidirectional degradation either from 5′ or 3′ termini, and detection of oligo(U) tracts from degradation products by either traditional or deep sequencing.