In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-Way System for RNAP II Transcription/Splicing/microRNA Processing.
In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-Way System for RNAP II Transcription/Splicing/microRNA Processing.
复制标题
用于将 RNAP II 转录与初级 microRNA 处理耦合的体外系统和用于 RNAP II 转录/剪接/microRNA 处理的三向系统。
DOI：
10.1007/978-1-4939-8624-8_4
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Reed,Robin
中科院分区：
文献类型：
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作者：
Yin,Shanye;Iocolano,Alexander;Yu,Yong;Gangopadhyay,Jaya;Reed,Robin
在基因组中，初级microrna （pri- mirna）被编码为独立的转录单位，其自身的启动子（基因间mirna）或在其他基因的内含子（内含子mirna）中编码。在这里，我们报告了两种方法，一种是我们建立的RNAP II转录和pri-miRNA加工的耦合方法，另一种是RNAP II转录、pri-miRNA加工和pre-mRNA剪接的三向系统。在这些系统中，编码加工底物的CMV-DNA构建体在32p - utp存在的情况下在HeLa细胞核提取物中孵育，产生新生的RNAP II转录本，这些转录本被核提取物中的内源性RNA加工机制有效地加工。
In the genome, primary microRNAs (pri-miRNAs) are encoded either as independent transcriptional units with their own promoters (intergenic miRNAs) or within the introns of other genes (intronic miRNAs). Here, we report two methods, one that we established for coupled RNAP II transcription and pri-miRNA processing and the other that is a three-way system for RNAP II transcription, pri-miRNA processing, and pre-mRNA splicing. In these systems, CMV-DNA constructs encoding the processing substrates are incubated in HeLa cell nuclear extracts in the presence of32P-UTP to generate the nascent RNAP II transcripts, which are processed efficiently by the endogenous RNA processing machineries in nuclear extracts.