Structural basis for the non-self RNA-activated protease activity of the type III-E CRISPR nuclease-protease Craspase

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• The RNA-targeting type III-E CRISPR-gRAMP effector forms a complex with TPR-CHAT protease, known as Craspase.
• The functional mechanism of Craspase in RNA targeting and protease activation remained largely unknown.

Purpose of the Study:

• To elucidate the structural basis and functional mechanism of the type III-E CRISPR-gRAMP-TPR-CHAT (Craspase) complex.
• To understand how Craspase discriminates between self and non-self RNA targets and activates protease activity.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structures of gRAMP-crRNA and gRAMP-crRNA-TPR-CHAT complexes.
• Structural analysis was performed before and after binding to self or non-self RNA targets.

Main Results:

• Cryo-EM structures revealed distinct TPR-CHAT conformations upon binding to self versus non-self RNA.
• Nucleotides at positions -1 and -2 of the crRNA act as sensors for target RNA identity.
• Non-self RNA binding specifically activated TPR-CHAT protease, leading to Csx30 cleavage.
• TPR-CHAT shares structural similarity with eukaryotic separase but has a unique regulatory mechanism.

Conclusions:

• The study reveals the mechanism of RNA-targeting and non-self RNA-induced protease activation by the type III-E CRISPR-Cas Craspase.
• Findings provide a foundation for developing gRAMP-based RNA manipulation tools.
• The research advances understanding of virus-host discrimination via the Craspase nuclease-protease system.