Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Brr2 is a DExD/H-box helicase essential for spliceosomal activation, specifically U4/U6 unwinding.
• Brr2 possesses two helicase-like domains and two Sec63 domains, with the latter's function being uncharacterized.
• Understanding Brr2's structure and mechanism is key to elucidating the intricacies of the splicing process.

Purpose of the Study:

• To determine the crystal structure of the second Sec63 domain of Brr2.
• To investigate the structural relationship between Brr2 and DNA helicase Hel308.
• To explore the functional interactions of Brr2 within the spliceosome, particularly with Prp8 and Snu114.

Main Methods:

• X-ray crystallography to determine the structure of Brr2's second Sec63 domain.
• Sequence analysis to compare Brr2 domains with known helicases, including Hel308.
• Site-directed mutagenesis and in vitro/in vivo interaction assays to study protein-protein binding and function.

Main Results:

• The crystal structure of Brr2's second Sec63 domain revealed unexpected resemblance to domains 4 and 5 of Hel308.
• Brr2 was hypothesized to contain two consecutive Hel308-like modules, suggesting a shared helicase mechanism.
• Brr2's Hel308-II module interacts with Prp8 and Snu114, and the Prp8 C-terminal region (Prp8-CTR) facilitates Brr2-Prp8-CTR binding to U4/U6.

Conclusions:

• Brr2 likely operates via a mechanism similar to Hel308, involving two consecutive Hel308-like modules.
• Brr2 interacts with key spliceosomal components Prp8 and Snu114, mediated by its Hel308-II module.
• The Prp8-CTR plays a crucial role in recruiting Brr2 to the U4/U6 snRNP, impacting spliceosomal activation regulation.