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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.

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In Vitro Transcription of Plant miRNA for Structural and Processing Analysis

  • 实验方案

  • 发表日期：

    2025年

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  • Corina Diaz-Quezada,
  • Noe Baruch-Torres,
  • Carlos H. Trasviña-Arenas,
  • Luis G. Brieba

摘要

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.