Henan Engineering Laboratory for Bioconversion Technology of Functional Microbes, College of Life Sciences, Henan Normal University, Xinxiang 453007, China.
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Int J Mol Sci. 2022 Jul 31;23(15):8515. doi: 10.3390/ijms23158515.
Type III CRISPR-Cas systems show the target (tg)RNA-activated indiscriminate DNA cleavage and synthesis of oligoadenylates (cOA) and a secondary signal that activates downstream nuclease effectors to exert indiscriminate RNA/DNA cleavage, and both activities are regulated in a spatiotemporal fashion. In III-B Cmr systems, cognate tgRNAs activate the two Cmr2-based activities, which are then inactivated via tgRNA cleavage by Cmr4, but how Cmr4 nuclease regulates the Cmr immunization remains to be experimentally characterized. Here, we conducted mutagenesis of Cmr4 conserved amino acids in , and this revealed that Cmr4α RNase-dead (dCmr4α) mutation yields cell dormancy/death. We also found that plasmid-borne expression of dCmr4α in the wild-type strain strongly reduced plasmid transformation efficiency, and deletion of CRISPR arrays in the host genome reversed the dCmr4α inhibition. Expression of dCmr4α also strongly inhibited plasmid transformation with Cmr2α and Cmr2α mutants, but the inhibition was diminished in Cmr2α. Since dCmr4α-containing effectors lack spatiotemporal regulation, this allows an everlasting interaction between crRNA and cellular RNAs to occur. As a result, some cellular RNAs, which are not effective in mediating immunity due to the presence of spatiotemporal regulation, trigger autoimmunity of the Cmr-α system in the cells expressing dCmr4α. Together, these results pinpoint the crucial importance of tgRNA cleavage in autoimmunity avoidance and in the regulation of immunization of type III systems.
III 型 CRISPR-Cas 系统表现出靶向（tg）RNA 激活的无差别 DNA 切割和寡聚腺苷酸（cOA）的合成，以及激活下游核酸酶效应物发挥无差别 RNA/DNA 切割的二级信号，这两种活性都受到时空调节。在 III-B Cmr 系统中，同源 tgRNA 激活基于两个 Cmr2 的活性，然后通过 Cmr4 切割 tgRNA 使其失活，但 Cmr4 核酸酶如何调节 Cmr 免疫仍有待实验表征。在这里，我们对 Cmr4 保守氨基酸进行了突变，结果显示 Cmr4α RNase 失活（dCmr4α）突变导致细胞休眠/死亡。我们还发现，野生型菌株中质粒携带的 dCmr4α表达强烈降低了质粒转化效率，而宿主基因组中 CRISPR 阵列的缺失则逆转了 dCmr4α 的抑制作用。dCmr4α 的表达也强烈抑制了 Cmr2α 和 Cmr2α 突变体的质粒转化，但在 Cmr2α 中抑制作用减弱。由于含有 dCmr4α 的效应物缺乏时空调节，这允许 crRNA 与细胞 RNA 之间发生永恒的相互作用。结果，一些由于时空调节而不能有效介导免疫的细胞 RNA，会在表达 dCmr4α 的细胞中引发 Cmr-α 系统的自身免疫。总之，这些结果指出了 tgRNA 切割在避免自身免疫和调节 III 型系统免疫中的重要性。
