Recognition and maturation of effector RNAs in a CRISPR interference pathway

Abstract

In bacteria and archaea, small RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci are involved in an adaptable and heritable gene-silencing pathway. Resistance to phage infection is conferred by the incorporation of short invading DNA sequences into the genome as CRISPR spacer elements separated by short repeat sequences. Processing of long primary transcripts (pre-crRNAs) containing these repeats by an RNA endonuclease generates the mature effector RNAs that interfere with phage gene expression. Here we describe structural and functional analyses of the Thermus thermophilus CRISPR Cse3 endonuclease. High-resolution X-ray structures of Cse3 bound to repeat RNAs model both the pre- and post-cleavage complexes associated with processing the pre-crRNA. These structures establish the molecular basis of a specific CRISPR RNA recognition and suggest the mechanism for generation of effector RNAs responsible for gene silencing.

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).…”