Laboratory of Molecular Developmental Biology, State Key Laboratory of Membrane Biology, Beijing 100084, China; Tsinghua-Peking Center for Life Sciences, Beijing 100084, China; School of Life Sciences, Tsinghua University, Beijing 100084, China.
Laboratory of Molecular Developmental Biology, State Key Laboratory of Membrane Biology, Beijing 100084, China; Tsinghua-Peking Center for Life Sciences, Beijing 100084, China; School of Life Sciences, Tsinghua University, Beijing 100084, China
Development. 2020 Nov 30;147(22):dev190728. doi: 10.1242/dev.190728.
Mini-III RNase (mR3), a member of RNase III endonuclease family, can bind to and cleave double-stranded RNAs (dsRNAs). Inactive mR3 protein without the α5β-α6 loop loses the dsRNA cleavage activity, but retains dsRNA binding activity. Here, we establish an inactive mR3-based non-engineered mR3/dsRNA system for RNA tracking in zebrafish embryos. binding experiments show that inactive mR3 (dSmR3) protein possesses the highest binding affinity with dsRNAs among mR3s from other related species, and its binding property is retained in zebrafish embryos. Combined with a fluorescein-labeled antisense RNA probe recognizing the target mRNAs, dSmR3 tagged with a nuclear localization sequence and a fluorescent protein could allow visualization of the dynamics of endogenous target mRNAs. The dSmR3/antisense probe dual-color system provides a new approach for tracking non-engineered RNAs in real-time, which will help understand how endogenous RNAs dynamically move during embryonic development.
Mini-III RNase（mR3）是 RNase III 内切核酸酶家族的成员，能够结合并切割双链 RNA（dsRNA）。没有 α5β-α6 环的无活性 mR3 蛋白失去了 dsRNA 切割活性，但保留了 dsRNA 结合活性。在这里，我们建立了一个基于无活性 mR3 的非工程 mR3/dsRNA 系统，用于追踪斑马鱼胚胎中的 RNA。结合实验表明，无活性的 mR3（dSmR3）蛋白在来自其他相关物种的 mR3 中与 dsRNA 具有最高的结合亲和力，并且其结合特性在斑马鱼胚胎中得以保留。结合带有荧光标记的针对靶 mRNA 的反义 RNA 探针，带有核定位序列和荧光蛋白的 dSmR3 可用于可视化内源性靶 mRNA 的动态。dSmR3/反义探针双色系统为实时追踪非工程 RNA 提供了一种新方法，这将有助于了解内源性 RNA 在胚胎发育过程中如何动态移动。
