Structural basis for conformational equilibrium of the catalytic spliceosome

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• The spliceosome is a dynamic molecular machine responsible for RNA splicing.
• The ATPase Prp16 plays a crucial role in regulating spliceosome conformation.
• Understanding spliceosome dynamics is key to deciphering gene expression regulation.

Purpose of the Study:

• To elucidate the molecular mechanism by which Prp16 governs spliceosome conformation.
• To identify novel spliceosome intermediates and their structural basis.
• To understand the role of Prp16 in stabilizing catalytic spliceosome conformations.

Main Methods:

• Electron cryomicroscopy (cryo-EM) reconstruction of the Saccharomyces cerevisiae C-complex spliceosome at 2.8 Å resolution.
• Biochemical assays to investigate factor binding and complex conversion.
• Structural analysis of spliceosome intermediates and complexes.

Main Results:

• A novel C-complex intermediate (Ci) was identified, revealing the molecular basis for spliceosome conformational equilibrium.
• Exon-ligation factors Prp18 and Slu7 bind to Ci before Prp16-mediated destabilization of the branching conformation.
• A complete model of the Prp19 complex (NTC) shows recruitment of branching factors Yju2 and Isy1, and how Prp16 remodels Yju2 binding for exon ligation.

Conclusions:

• Prp16 action modulates the dynamic binding of step-specific factors to stabilize C or C* conformations.
• The identified intermediate and factor interactions explain how Prp16 establishes equilibrium of the catalytic spliceosome.
• This study provides a detailed molecular understanding of spliceosome dynamics and regulation.