Csb1 moonlighting gives rise to functional redundancy with Csb2 in processing the pre-CRISPR transcript in type I-G CRISPR-Cas system

Abstract

Archaea and bacteria use CRISPR-based adaptive immunity to limit the genome invasion by phages. Among the type-I CRISPR variant, the newly discovered type I-G exhibits unusual variation in the composition and architecture of Cas proteins. In order to understand how these structural differences, contribute to functional adaptations, we probed how the maturation of CRISPR RNA differs with respect to other well studied type I CRISPR variants. Type I-G consists of three Cas proteins, viz, Csb1, Csb2 and Csb3 that are predicted to form the ribonucleoprotein surveillance effector complex. We show that Csb2 from Bifidobacterium animalis is a metal independent endonuclease that cleaves site-specifically within the 5 region of the CRISPR repeat RNA. The catalytic activity resides within the C-terminal region that is homologous to Cas6. Interestingly, Csb2 processes the pre-CRISPR transcript both as a stand-alone enzyme and as a subunit of the Cascade/I-G complex that comprises of Csb1, Csb2 and Csb3 in association with crRNA. Surprisingly, we discovered that Csb1-which is homologous to Cas7 that is catalytically inert in other type I systems-also shows metal independent RNase activity that is functionally analogous to Csb2 in processing the pre-CRISPR RNA. The presence of dual nucleases in the Cascade/I-G complex enhances the efficiency of CRISPR-based immunity. We suggest that the Csb1 moonlighting engenders functional redundancy between Csb1 and Csb2 that in turn could compensate for the intrinsic instability of Csb2 and accelerate the maturation of crRNA.