Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA.
Department of Chemistry, University of Alabama, Tuscaloosa, AL, 35487, USA.
Nucleic Acids Res. 2017 Feb 28;45(4):2112-2123. doi: 10.1093/nar/gkw891.
CRISPR–Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a prokaryotic immune system that destroys foreign nucleic acids in a sequence-specific manner using Cas nucleases guided by short RNAs (crRNAs). Staphylococcus epidermidis harbours a Type III-A CRISPR–Cas system that encodes the Cas10–Csm interference complex and crRNAs that are subjected to multiple processing steps. The final step, called maturation, involves a concerted effort between Csm3, a ruler protein in Cas10–Csm that measures six-nucleotide increments, and the activity of a nuclease(s) that remains unknown. Here, we elucidate the contributions of the Cas10–Csm complex toward maturation and explore roles of non-Cas nucleases in this process. Using genetic and biochemical approaches, we show that charged residues in Csm3 facilitate its self-assembly and dictate the extent of maturation cleavage. Additionally, acidic residues in Csm5 are required for efficient maturation, but recombinant Csm5 fails to cleave crRNAs in vitro. However, we detected cellular nucleases that co-purify with Cas10–Csm, and show that Csm5 regulates their activities through distinct mechanisms. Altogether, our results support roles for non-Cas nuclease(s) during crRNA maturation and establish a link between Type III-A CRISPR–Cas immunity and central nucleic acid metabolism.
CRISPR–Cas（成簇规律间隔短回文重复序列-CRISPR 相关蛋白）是一种原核免疫系统，它使用 Cas 核酸酶以序列特异性方式破坏外来核酸，这些 Cas 核酸酶由短 RNA（crRNA）引导。表皮葡萄球菌含有一种 III-A 型 CRISPR–Cas 系统，该系统编码 Cas10–Csm 干扰复合物和 crRNA，这些 crRNA 经历多个加工步骤。最后一步称为成熟，涉及 Cas10–Csm 中的尺子蛋白 Csm3 和活性未知的核酸酶之间的协同作用。在这里，我们阐明了 Cas10–Csm 复合物对成熟的贡献，并探讨了非 Cas 核酸酶在这个过程中的作用。通过遗传和生化方法，我们表明 Csm3 中的带电残基促进其自组装并决定成熟切割的程度。此外，Csm5 中的酸性残基对于有效的成熟是必需的，但重组 Csm5 不能在体外切割 crRNA。然而，我们检测到与 Cas10–Csm 共纯化的细胞核酸酶，并表明 Csm5 通过不同的机制调节它们的活性。总之，我们的结果支持非 Cas 核酸酶（s）在 crRNA 成熟过程中的作用，并建立了 III-A 型 CRISPR–Cas 免疫与中心核酸代谢之间的联系。
