Shanghai Public Health Clinical Center, State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200438, China.
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200438, China.
Nucleic Acids Res. 2021 Jan 11;49(1):568-583. doi: 10.1093/nar/gkaa1197.
Infection with kinetoplastid parasites, including Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania can cause serious disease in humans. Like other kinetoplastid species, mRNAs of these disease-causing parasites must undergo posttranscriptional editing in order to be functional. mRNA editing is directed by gRNAs, a large group of small RNAs. Similar to mRNAs, gRNAs are also precisely regulated. In T. brucei, overexpression of RNase D ribonuclease (TbRND) leads to substantial reduction in the total gRNA population and subsequent inhibition of mRNA editing. However, the mechanisms regulating gRNA binding and cleavage by TbRND are not well defined. Here, we report a thorough structural study of TbRND. Besides Apo- and NMP-bound structures, we also solved one TbRND structure in complexed with single-stranded RNA. In combination with mutagenesis and in vitro cleavage assays, our structures indicated that TbRND follows the conserved two-cation-assisted mechanism in catalysis. TbRND is a unique RND member, as it contains a ZFD domain at its C-terminus. In addition to T. brucei, our studies also advanced our understanding on the potential gRNA degradation pathway in T. cruzi, Leishmania, as well for as other disease-associated parasites expressing ZFD-containing RNDs.
动基体原生动物寄生虫（包括布氏锥虫（Trypanosoma brucei）、克氏锥虫（Trypanosoma cruzi）和利什曼原虫）感染可导致人类严重疾病。与其他动基体原生动物物种一样，这些致病寄生虫的 mRNA 必须经过转录后编辑才能发挥功能。mRNA 编辑由 gRNA 指导，gRNA 是一大组小 RNA。与 mRNAs 相似，gRNAs 也受到精确调控。在布氏锥虫中，核糖核酸酶 D（TbRND）的过度表达会导致总 gRNA 群体大量减少，并随后抑制 mRNA 编辑。然而，调节 gRNA 与 TbRND 结合和切割的机制尚未得到很好的定义。在这里，我们报告了对 TbRND 的全面结构研究。除了 apo 和 NMP 结合结构外，我们还解决了一个与单链 RNA 复合物的 TbRND 结构。结合突变和体外切割实验，我们的结构表明 TbRND 在催化中遵循保守的双阳离子辅助机制。TbRND 是一种独特的 RND 成员，因为它在 C 末端含有一个 ZFD 结构域。除了布氏锥虫，我们的研究还增进了对克氏锥虫、利什曼原虫以及其他表达含 ZFD 的 RND 的与疾病相关寄生虫中潜在 gRNA 降解途径的理解。
