An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR) chromosomal loci found in prokaryotes provide an adaptive immune system against bacteriophages and plasmids. CRISPR-specific endoRNases produce short RNA molecules (crRNAs) from CRISPR transcripts, which harbor sequences complementary to invasive nucleic acid elements and ensure their selective targeting by CRISPR-associated (Cas) proteins. The extreme sequence divergence of CRISPR-specific endoRNases and their RNA substrates has obscured homology-based comparison of RNA recognition and cleavage mechanisms. Here, we show that Cse3 type CRISPR-specific endoRNases bind a hairpin structure and residues downstream of the cleavage site within the repetitive segment of cognate CRISPR RNA. Cocrystal structures of Cse3-RNA complexes reveal an RNA-induced conformational change in the enzyme active site that aligns the RNA strand for site-specific cleavage. These studies provide insight into a catalytically essential RNA recognition mechanism by a large class of CRISPR-related endoRNases.

The impact of CRISPR repeat sequence on structures of a Cas6 protein–RNA complex

“…20 The recently determined co-crystal structures of Cas6, Csy4, and Cse3 indeed revealed two different modes of RNA interactions. 18,[21][22][23] Cas6 specifically binds to the 5 0 terminus of its single-stranded substrates 21 while Csy4 and Cse3 bind to a stable hairpin immediately preceding the cleavage site. 18,22,23 These results suggest that CRISPR processing machineries must recognize structurally diverse pre-crRNA and likely coevolves with their CRISPR RNA substrates.…”

“…18,[21][22][23] Cas6 specifically binds to the 5 0 terminus of its single-stranded substrates 21 while Csy4 and Cse3 bind to a stable hairpin immediately preceding the cleavage site. 18,22,23 These results suggest that CRISPR processing machineries must recognize structurally diverse pre-crRNA and likely coevolves with their CRISPR RNA substrates. The molecular basis for specific interaction between the processing endonucleases and their respective substrates can help us understand the remarkable diversity among RAMP proteins.…”

Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site

“…In Type III systems, crRNA intermediates are subject to additional nucleolytic attack at the 3′ end, generating mature crRNAs with reduced length (18,19). This final maturation step seems to be absent in Type I systems (10,12,13,15). In Type II systems, a trans-encoded crRNA complementary to the precursor crRNA guides primary processing.…”

“…Repeat spacer arrays and their adjacent cas genes are classified into three CRISPR/Cas types (I-III) (17) that undergo different mechanisms of crRNA processing. In Types I and III CRISPR/Cas systems, primary processing is achieved by Cas6 endoribonucleases and results in crRNA intermediates with 5′-hydroxyl and 3′-phosphate or 2′-3′-cyclic phosphate ends (9,10,12,13). Cleavage occurs 8 nt upstream of the beginning of the spacer sequence, leaving a 5′ handle (6) or crRNA tag (18) on the 5′ end.…”

“…CRISPR arrays are transcribed into a long precursor containing spacers and repeats that are processed into small CRISPR RNAs (crRNAs) by dedicated CRISPR-associated (Cas) endoribonucleases (6,8,9). crRNAs act as guides for a targeting complex (10)(11)(12)(13)(14)(15) that cleaves the genetic material of the invading bacteriophage or plasmid (16).…”