Clustered regularly interspaced short palindromic repeats (CRISPRs) confer
adaptive immunity to prokaryotes through a small RNA-mediated mechanism.
Specific endoribonucleases are required by all CRISPR-bearing organisms to
process CRISPR RNAs into small RNA that serve as guides for defensive effector
complexes. The molecular mechanism of how the endoribonucleases process the
class of CRISPR RNA containing no predicted secondary structural features
remains largely elusive. Here, we report cocrystal structures of a processing
endoribonuclease bound with a noncleavable RNA substrate and its product-like
fragment derived from a nonpalindramic repeat. The enzyme stabilizes a short RNA
stem-loop structure near the cleavage site and cleaves the phosphodiester bond
using an active site comprised of arginine and lysine residues. The distinct RNA
binding and cleavage mechanisms underline the diversity in CRISPR RNA processing.
