Investigation of the mechanism of Brr2-mediated dissociation of U4/U6 di-snRNAs

Brr2 is the largest RNA helicase and a unique representative of Ski2-like subfamily of DExD/H box proteins in the spliceosome. It contains two tandem putative helicase cassettes; each encompasses a dual RecA-like domain and a Sec63 unit. In process of the spliceosome activation, Brr2 unwinds the long base pairing between U4 and U6 di-snRNAs which leads to release of U4. This makes U6 free for adopting an alternative conformation promoting splicing catalysis. A recent crystal structure of the C-terminal Sec63 unit of Brr2 showed unexpected structural similarity of two domains to functional modules of Hel308, a processive DNA helicase which targets fork DNA substrates. We have validated the modelled Brr2 structure by mutagenesis and conducing U4/U6 RNA unwinding assays. We provide evidence that Brr2 binds to U4/U6 in the absence of ATP and that it requires single-stranded overhangs of U4/U6 to be loaded onto this RNA duplex. In addition, Brr2 showed a lower unwinding activity towards linear RNA duplex substrates. Within the U5 snRNP, Brr2 is tightly associated to Prp8 and its time of action is regulated through a combinatorial function of Prp8 and the EF-G-like GTPase Snu114. Of our particular interest is the C-terminal portion of Prp8. This region consists of an RNase H-like domain and a Jab1/MPN-like domain which is followed by a C-terminal tail extending out of the Jab1 structure. Several mutations within this tail are linked to autosomal dominant retinitis pigmentosa (RP13), a progressive retinal dystrophy. Here, we have investigated the regulatory function of Prp8 C-terminal fragments on Brr2. In addition, we have examined the effect of RP13-linked missense or nonsense mutations in the regulation of Brr2-catalyzed unwinding of U4/U6.