Multimeric assembly and biochemical characterization of the Trax-translin endonuclease complex

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Trax-translin heteromers (C3PO) are implicated in RNA interference.
• C3PO is proposed to activate RISC by cleaving siRNA passenger strands.

Purpose of the Study:

• To determine the structure and function of Drosophila C3PO.
• To elucidate the role of Trax in C3PO assembly and activity.

Main Methods:

• X-ray crystallography of hexameric C3PO.
• Electron microscopy and mass spectrometry of octameric C3PO.
• Biochemical assays to measure endoRNase activity.

Main Results:

• Crystal structure of hexameric and electron microscopy of octameric C3PO determined.
• Trax adopts the translin fold and contains catalytic centers.
• C3PO exhibits high endoRNase activity, cleaving substrate at stoichiometric rates.
• Catalytic pockets are located in the interior of the octameric assembly.

Conclusions:

• Trax is essential for C3PO assembly and possesses the catalytic activity.
• C3PO functions as an endoRNase, cleaving siRNA passenger strands to activate RISC.
• The structural and biochemical data provide mechanistic insights into C3PO function.