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Keywords:

  • CRISPR-Cas;
  • RAMP proteins;
  • protein–RNA interactions;
  • RRM fold;
  • RNA-mediated prokaryotic immunity

Abstract

The repeat-associated mysterious proteins (RAMPs) comprise the most abundant family of proteins involved in prokaryotic immunity against invading genetic elements conferred by the clustered regularly interspaced short palindromic repeat (CRISPR) system. Cas6 is one of the first characterized RAMP proteins and is a key enzyme required for CRISPR RNA maturation. Despite a strong structural homology with other RAMP proteins that bind hairpin RNA, Cas6 distinctly recognizes single-stranded RNA. Previous structural and biochemical studies show that Cas6 captures the 5′ end while cleaving the 3′ end of the CRISPR RNA. Here, we describe three structures and complementary biochemical analysis of a noncatalytic Cas6 homolog from Pyrococcus horikoshii bound to CRISPR repeat RNA of different sequences. Our study confirms the specificity of the Cas6 protein for single-stranded RNA and further reveals the importance of the bases at Positions 5–7 in Cas6–RNA interactions. Substitutions of these bases result in structural changes in the protein–RNA complex including its oligomerization state.