Essential Structural and Functional Roles of the Cmr4 Subunit in RNA Cleavage by the Cmr CRISPR-Cas Complex
Nancy F. Ramia,
Michael Spilman,
Li Tang,
Yaming Shao,
Joshua Elmore,
Caryn Hale,
Alexis Cocozaki,
Nilakshee Bhattacharya,
Rebecca M. Terns,
Michael P. Terns,
Hong Li,
Scott M. Stagg
Affiliations
Nancy F. Ramia
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA
Michael Spilman
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA
Li Tang
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA
Yaming Shao
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA
Joshua Elmore
Departments of Biochemistry and Molecular Biology, Genetics, and Microbiology, University of Georgia, Athens, GA 30602, USA
Caryn Hale
Departments of Biochemistry and Molecular Biology, Genetics, and Microbiology, University of Georgia, Athens, GA 30602, USA
Alexis Cocozaki
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA
Nilakshee Bhattacharya
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA
Rebecca M. Terns
Departments of Biochemistry and Molecular Biology, Genetics, and Microbiology, University of Georgia, Athens, GA 30602, USA
Michael P. Terns
Departments of Biochemistry and Molecular Biology, Genetics, and Microbiology, University of Georgia, Athens, GA 30602, USA; Corresponding author
Hong Li
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA; Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA; Corresponding author
Scott M. Stagg
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA; Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA; Corresponding author
Summary: The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA) and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes. : Ramia et al. show that the helical core of the type III-B Cmr CRISPR-Cas effector complex, made up of multiple Cmr4 subunits, forms the platform for a corresponding number of cleavages of the target RNA. Comparison with the type I-E Cascade structure reveals strikingly similar mechanisms of crRNA and target binding.
