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The host-encoded RNase E endonuclease as the crRNA maturation enzyme in a CRISPR–Cas subtype III-Bv system


Specialized RNA endonucleases for the maturation of clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNAs (crRNAs) are critical in CRISPR–CRISPR-associated protein (Cas) defence mechanisms. The Cas6 and Cas5d enzymes are the RNA endonucleases in many class 1 CRISPR–Cas systems. In some class 2 systems, maturation and effector functions are combined within a single enzyme or maturation proceeds through the combined actions of RNase III and trans-activating CRISPR RNAs (tracrRNAs). Three separate CRISPR–Cas systems exist in the cyanobacterium Synechocystis sp. PCC 6803. Whereas Cas6-type enzymes act in two of these systems, the third, which is classified as subtype III-B variant (III-Bv), lacks cas6 homologues. Instead, the maturation of crRNAs proceeds through the activity of endoribonuclease E, leaving unusual 13- and 14-nucleotide-long 5′-handles. Overexpression of RNase E leads to overaccumulation and knock-down to the reduced accumulation of crRNAs in vivo, suggesting that RNase E is the limiting factor for CRISPR complex formation. Recognition by RNase E depends on a stem-loop in the CRISPR repeat, whereas base substitutions at the cleavage site trigger the appearance of secondary products, consistent with a two-step recognition and cleavage mechanism. These results suggest the adaptation of an otherwise very conserved housekeeping enzyme to accommodate new substrates and illuminate the impressive plasticity of CRISPR–Cas systems that enables them to function in particular genomic environments.

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We thank O. Alkhnbashi and C. Steglich for helpful comments, U. Ruppert for technical assistance, M. Asayama and E. P. Hudson for the provided plasmids. Financial support from the German Research Foundation programme FOR1680 'Unravelling the Prokaryotic Immune System' (grants HE 2544/8-2 and UR225/1-2) to W.R.H. and H.U. is greatly acknowledged.

Author information

J.B. and W.R.H. designed the work. The protein–RNA cross-linking experiments and identification of cross-linked peptide–RNA bonds were performed by K.S. and H.U. The RNase E overexpression and partial deletion strains were constructed by A.W. The ∆C1 and ∆C1∆C2 deletion strains were constructed by V.R. All other experiments were carried out by J.B. J.B., K.S. and W.R.H. analysed the data. J.B. and W.R.H. wrote the paper with contributions from all authors.

Correspondence to Wolfgang R. Hess.

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Supplementary Figures 1–6, Supplementary Table 1, raw full length gels and blots.

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Fig. 1: Organization of Synechocystis 6803 CRISPR3 cas genes, repeat-spacer array and interference assay.
Fig. 2: Mapping of in vivo processing sites within CRISPR3 pre-crRNA by primer extension.
Fig. 3: RNase E substrates and cleavage sites in Synechocystis 6803.
Fig. 4: Manipulation of RNase E expression affects CRISPR3 crRNA accumulation.
Fig. 5: Identification and analysis of RNase E amino acids interacting with CRISPR3 repeat RNA.
Fig. 6: Model for RNase-E-mediated crRNA recognition and processing.