Laboratoire Génome et Développement des Plantes, UMR 5096, CNRS - Université Perpignan Via Domitia, Perpignan, France.
Methods Mol Biol. 2021;2209:363-385. doi: 10.1007/978-1-0716-0935-4_23.
RTL (RNase three-like) proteins belong to a distinct family of endonucleases that cleave double-stranded RNAs in plants. RTL1 to 3 are structurally related to the RNAse III from E. coli and formally belong to the class 1 of RNase III proteins. RTLs have conserved RNase III signature motif(s) and up to two dsRNA binding (DRB) domains. RTLs target and cleave coding and noncoding dsRNAs, including precursors of ribosomal (rRNA), small interference (siRNA), and micro (miRNA) RNAs. Interestingly, RTL proteins have stronger affinity than RNase III-Dicer proteins for dsRNA precursors of siRNAs, but not for miRNAs. However, very little is known of the structural and molecular bases directing and controlling RTL-RNA binding and activity. To address these questions, we have developed in vitro cleavage assays that combine recombinant RTL1 protein and in vitro transcribed or plant-extracted RNAs, RT-PCR, and primer extension experiments or analysis.
RTL（核糖核酸酶 III 样）蛋白属于一类在植物中切割双链 RNA 的内切核酸酶，与大肠杆菌中的 RNAse III 结构相关，正式属于 RNAse III 蛋白的第 1 类。RTL 具有保守的 RNAse III 特征基序和多达两个双链 RNA 结合（DRB）结构域。RTL 靶向并切割编码和非编码的双链 RNA，包括核糖体（rRNA）、小干扰（siRNA）和微（miRNA）RNA 的前体。有趣的是，与 RNase III-Dicer 蛋白相比，RTL 蛋白对 siRNA 的 dsRNA 前体具有更强的亲和力，但对 miRNA 则不然。然而，对于指导和控制 RTL-RNA 结合和活性的结构和分子基础，我们知之甚少。为了解决这些问题，我们开发了体外切割测定法，该方法结合了重组 RTL1 蛋白和体外转录或植物提取的 RNA、RT-PCR 和引物延伸实验或分析。
