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

Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding, a critical step in spliceosomal activation. Brr2 contains an N-terminal domain and two tandem sets of a helicase-like domain followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. The helicase-like domain upstream of Sec63 has clear sequence similarity with domains 1-3 of Hel308. In addition modeling indicates that Brr2 is composed of an N-terminal domain and two consecutive Hel308-like modules (Hel308-I and II). Together this provides our first glimpse of the overall structure of this large and unique spliceosomal ATPase and helicase. Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism to Hel308, that differs from many DEAD-box proteins. We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo, potentially serving as a mediator for the regulation of Brr2’s activity by Prp8. We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2/Prp8-CTR complex to U4/U6, suggesting a potential role of Prp8-CTR as an auxiliary substrate binding and specificity domain for Brr2. Splicing specific microarrays were used to assess the genome-wide defects in pre-mRNA splicing that result from a deletion of the second Sec63 domain of yeast Brr2.