Two Distant Catalytic Sites Are Responsible for C2c2 RNase Activities

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Type VI CRISPR-Cas systems utilize the C2c2 effector protein for RNA-guided DNA or RNA interference.
• C2c2 exhibits two distinct RNase activities: one for target RNA cleavage and another for CRISPR RNA (crRNA) processing.

Purpose of the Study:

• To elucidate the structural basis of C2c2's dual RNase activities.
• To understand the mechanism of crRNA binding and its role in C2c2 function.

Main Methods:

• X-ray crystallography was employed to determine the structures of Leptotrichia shahii C2c2 in both crRNA-free and crRNA-bound states.
• Comparative structural analysis was performed to identify key domains and catalytic sites.

Main Results:

• The structures reveal a bilobed C2c2 protein with REC and NUC lobes, containing Helical-1 and two HEPN domains, respectively.
• Independent catalytic pockets for pre-crRNA processing (Helical-1) and target RNA cleavage (HEPN) were identified.
• crRNA binding induces significant conformational changes, enhancing crRNA stability and facilitating target RNA recognition.

Conclusions:

• The determined structures provide critical insights into the molecular mechanisms underlying C2c2's dual RNase activities.
• These findings lay the groundwork for engineering C2c2 as a novel RNA editing tool.