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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.

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Figure 1: Structure of Cse3 bound to SL+2 RNA at 1.8-Å resolution.
Figure 2: The active site of Cse3 with RNA bound.
Figure 3: Structures of Cse3 bound to SL+1 RNA.
Figure 4: Single-turnover cleavage analysis of crRNA or Cse3 mutants.
Figure 5: Binding of crRNA and Cse3 mutants.
Figure 6: Multiple-turnover cleavage analysis of crRNA and Cse3 mutants.
Figure 7: Comparison of Cse3 structure with Cas6 and Csy4.

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Acknowledgements

We thank E. Underbakke (UC Berkeley) for protein mass spectrometry; B. Wiedenheft, R. Haurwitz, S. Sternberg, K. Berry (UC Berkeley) and S. Coyle (UCSF) for helpful discussions; members of the Doudna laboratory for critical reading of the manuscript; and C. Ralston and J. Holton (Beamlines 8.2.1 and 8.3.1, Advanced Light Source, Lawrence Berkeley National Laboratory) for assistance with X-ray data collection. D.G.S. is supported by a Damon Runyon Cancer Research Foundation fellowship. M.J. is supported by a Human Frontier Science Program Long-Term Fellowship. This work was supported in part by grants from the National Science Foundation and the Bill and Melinda Gates Foundation to J.A.D. J.A.D. is a Howard Hughes Medical Institute investigator.

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D.G.S. and J.A.D. designed experiments. D.G.S. did all assays, crystallized the Cse3–RNA complexes and determined their structures. M.J. assisted with X-ray data collection and structure determination. D.G.S. and J.A.D. wrote the manuscript.

Corresponding author

Correspondence to Jennifer A Doudna.

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The authors declare no competing financial interests.

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Sashital, D., Jinek, M. & Doudna, J. An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3. Nat Struct Mol Biol 18, 680–687 (2011). https://doi.org/10.1038/nsmb.2043

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