Danish Archaea Center, Department of Biology, University of Copenhagen , Copenhagen N , Denmark.
State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China.
RNA Biol. 2019 Oct;16(10):1513-1520. doi: 10.1080/15476286.2019.1642725. Epub 2019 Jul 17.
Type III CRISPR-Cas systems code for a multi-subunit ribonucleoprotein (RNP) complex that mediates DNA cleavage and synthesizes cyclic oligoadenylate (cOA) second messenger to confer anti-viral immunity. Both immune activities are to be activated upon binding to target RNA transcripts by their complementarity to crRNA, and autoimmunity avoidance is determined by extended complementarity between the 5'-repeat tag of crRNA and 3'-flanking sequences of target transcripts (anti-tag). However, as to how the strategy could achieve stringent autoimmunity avoidance remained elusive. In this study, we systematically investigated how the complementarity of the crRNA 5'-tag and anti-tag (i.e., tag complementarity) could affect the interference activities (DNA cleavage activity and cOA synthesis activity) of Cmr-α, a type III-B system in Rey15A. The results revealed an increasing suppression on both activities by increasing degrees of tag complementarity and a critical function of the 7 nucleotide of crRNA in avoiding autoimmunity. More importantly, mutagenesis of Cmr3α exerts either positive or negative effects on the cOA synthesis activity depending on the degrees of tag complementarity, suggesting that the subunit, coupling with the interaction between crRNA tag and anti-tag, function in facilitating immunity and avoiding autoimmunity in Type III-B systems.
III 型 CRISPR-Cas 系统编码一个多亚基核糖核蛋白（RNP）复合物，介导 DNA 切割，并合成环状寡腺苷酸（cOA）第二信使，从而赋予抗病毒免疫。这两种免疫活性都是在通过与互补的 crRNA 结合到靶 RNA 转录本上时被激活的，而自身免疫的避免则是由 crRNA 的 5'-重复标签和靶转录本的 3'-侧翼序列（抗标签）之间的扩展互补性决定的。然而，关于这种策略如何能够实现严格的自身免疫避免，仍然难以捉摸。在这项研究中，我们系统地研究了 crRNA 5'-标签和抗标签（即标签互补性）的互补性如何影响 Cmr-α的干扰活性（DNA 切割活性和 cOA 合成活性），Cmr-α 是 Rey15A 中的 III-B 系统。结果表明，随着标签互补性的增加，两种活性都受到越来越大的抑制，并且 crRNA 的 7 个核苷酸在避免自身免疫方面具有关键功能。更重要的是，Cmr3α 的突变对 cOA 合成活性产生积极或消极的影响，这取决于标签互补性的程度，这表明该亚基与 crRNA 标签和抗标签之间的相互作用一起，在 III-B 型系统中促进免疫和避免自身免疫。
