Crystal structures of CRISPR-associated Csx3 reveal a manganese-dependent deadenylation exoribonuclease

ABSTRACTIn prokaryotes, the CRISPR/Cas system is known to target and degrade invading phages and foreign genetic elements upon subsequent infection. However, the structure and function of many Cas proteins remain largely unknown, due to the high diversity of Cas proteins. Here we report three crystal structures of <i>Archaeoglobus fulgidus</i> Csx3 (<i>Af</i>Csx3) in free form, in complex with manganese ions and in complex with a single-stranded RNA (ssRNA) fragment, respectively. <i>Af</i>Csx3 harbors a ferredoxin-like fold and forms dimer both in the crystal and in solution. Our structure-based biochemical analysis demonstrates that the RNA binding sites and cleavage sites are located at two separate surfaces within the <i>Af</i>Csx3 dimer, suggesting a model to bind, tether and cleave the incoming RNA substrate. In addition, <i>Af</i>Csx3 displays robust 3′-deadenylase activity in the presence of manganese ions, which strongly suggests that <i>Af</i>Csx3 functions as a deadenylation exonuclease. Taken together, our results indicate that <i>Af</i>Csx3 is a Cas protein involved in RNA deadenylation and provide a framework for understanding the role of <i>Af</i>Csx3 in the Type III-B CRISPR/Cas system.