用于结构和加工分析的植物微小RNA的体外转录
In Vitro Transcription of Plant miRNA for Structural and Processing Analysis.
作者信息
Diaz-Quezada Corina, Baruch-Torres Noe, Trasviña-Arenas Carlos H, Brieba Luis G
机构信息
Unidad de Genomica Avanzada (UGA), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav), Carretera Irapuato-León, Irapuato, Guanajuato, Mexico.
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, USA Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX, USA.
出版信息
Methods Mol Biol. 2025;2900:107-116. doi: 10.1007/978-1-0716-4398-3_6.
Plant miRNAs are short gene repressors, ranging in length from 20 to 24 nucleotides, that specifically target mRNAs to effectively promote cleavage or impinge translation. Long primary miRNAs (pri-miRNA) from plants fold into double-stranded RNA structures with stem loop structures. These structures are then processed by an RNase type III enzyme and several other proteins. A functional mature miRNA selects its mRNA target through complementary base pairing. Importantly, plant pri-miRNA are variable in size and structure. Here, we describe a protocol to identify plant pri-miRNA processing intermediates by in vitro T7 RNA polymerase transcription and its purification for enzymatic and chemical footprinting as a tool to analyze the peculiarities of plant miRNA processing.
植物微小RNA（miRNA）是短基因阻遏物，长度在20到24个核苷酸之间，它们特异性靶向信使核糖核酸（mRNA）以有效促进切割或影响翻译。植物的长初级miRNA（pri-miRNA）折叠成双链RNA结构，带有茎环结构。这些结构随后由一种III型核糖核酸酶（RNase）和其他几种蛋白质进行加工。功能性成熟miRNA通过互补碱基配对选择其mRNA靶标。重要的是，植物pri-miRNA在大小和结构上是可变的。在这里，我们描述了一种通过体外T7 RNA聚合酶转录来鉴定植物pri-miRNA加工中间体并对其进行纯化以用于酶促和化学足迹分析的方法，以此作为分析植物miRNA加工特性的工具。
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