Structural basis of catalytic activation in human splicing

Area of Science:

• Molecular Biology
• RNA Splicing Mechanisms
• Protein-RNA Interactions

Background:

• Pre-mRNA splicing is a critical cellular process regulated by ATP-dependent RNA helicases.
• Catalytic activation of the spliceosome, a key step, involves remodeling mediated by the helicase PRP2 (DHX16).
• The precise function of PRP2 and the involvement of other helicases in this process remained unclear.

Purpose of the Study:

• To elucidate the mechanism of ATP-dependent spliceosome catalytic activation.
• To investigate the roles of RNA helicases PRP2 and Aquarius in spliceosome remodeling.
• To determine the structural basis of spliceosome activation using cryogenic electron microscopy.

Main Methods:

• Cryogenic electron microscopy (cryo-EM) to determine the structure of spliceosome intermediates.
• Biochemical assays to analyze the activity of helicases PRP2 and Aquarius.
• Genetic manipulation to study the effects of helicase inactivation on splicing.

Main Results:

• Catalytic activation occurs in two distinct ATP-dependent stages, driven by PRP2 and Aquarius.
• The cryo-EM structure of the B^AQR complex reveals how PRP2 and Aquarius remodel the spliceosome.
• Aquarius facilitates PRP2 dissociation and the relocation of the branch duplex, enabling catalysis.

Conclusions:

• This study reveals a two-stage model for spliceosome catalytic activation involving PRP2 and Aquarius.
• It provides a structural and mechanistic understanding of how these DEAH helicases coordinate their functions.
• The findings offer a paradigm for understanding coordinated helicase activity in complex molecular machines.