Yin Shanye, Iocolano Alexander, Yu Yong, Gangopadhyay Jaya, Reed Robin
Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
Methods Mol Biol. 2018;1823:43-50. doi: 10.1007/978-1-4939-8624-8_4.
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 of P-UTP to generate the nascent RNAP II transcripts, which are processed efficiently by the endogenous RNA processing machineries in nuclear extracts.
在基因组中，初级微小RNA（pri-miRNA）要么作为具有自身启动子的独立转录单元进行编码（基因间miRNA），要么位于其他基因的内含子中（内含子miRNA）。在此，我们报告了两种方法，一种是我们建立的用于RNA聚合酶II（RNAP II）转录与pri-miRNA加工偶联的方法，另一种是用于RNAP II转录、pri-miRNA加工和前体mRNA剪接的三向系统。在这些系统中，编码加工底物的巨细胞病毒（CMV）-DNA构建体在含有P-UTP的HeLa细胞核提取物中孵育，以生成新生的RNAP II转录本，这些转录本可被核提取物中的内源性RNA加工机制有效加工。
