Structural basis for dual roles of Aar2p in U5 snRNP assembly.

Yeast U5 small nuclear ribonucleoprotein particle (snRNP) is assembled via a cytoplasmic precursor that contains the U5-specific Prp8 protein but lacks the U5-specific Brr2 helicase. Instead, pre-U5 snRNP includes the Aar2 protein not found in mature U5 snRNP or spliceosomes. Aar2p and Brr2p bind competitively to a C-terminal region of Prp8p that comprises consecutive RNase H-like and Jab1/MPN-like domains. To elucidate the molecular basis for this competition, we determined the crystal structure of Aar2p in complex with the Prp8p RNase H and Jab1/MPN domains. Aar2p binds on one side of the RNase H domain and extends its C terminus to the other side, where the Jab1/MPN domain is docked onto a composite Aar2p-RNase H platform. Known Brr2p interaction sites of the Jab1/MPN domain remain available, suggesting that Aar2p-mediated compaction of the Prp8p domains sterically interferes with Brr2p binding. Moreover, Aar2p occupies known RNA-binding sites of the RNase H domain, and Aar2p interferes with binding of U4/U6 di-snRNA to the Prp8p C-terminal region. Structural and functional analyses of phospho-mimetic mutations reveal how phosphorylation reduces affinity of Aar2p for Prp8p and allows Brr2p and U4/U6 binding. Our results show how Aar2p regulates both protein and RNA binding to Prp8p during U5 snRNP assembly.

Structural basis for functional cooperation between tandem helicase cassettes in Brr2-mediated remodeling of the spliceosome.

Assembly of a spliceosome, catalyzing precursor-messenger RNA splicing, involves multiple RNA-protein remodeling steps, driven by eight conserved DEXD/H-box RNA helicases. The 250-kDa Brr2 enzyme, which is essential for U4/U6 di-small nuclear ribonucleoprotein disruption during spliceosome catalytic activation and for spliceosome disassembly, is the only member of this group that is permanently associated with the spliceosome, thus requiring its faithful regulation. At the same time, Brr2 represents a unique subclass of superfamily 2 nucleic acid helicases, containing tandem helicase cassettes. Presently, the mechanistic and regulatory consequences of this unconventional architecture are unknown. Here we show that in human Brr2, two ring-like helicase cassettes intimately interact and functionally cooperate and how retinitis pigmentosa-linked Brr2 mutations interfere with the enzyme's function. Only the N-terminal cassette harbors ATPase and helicase activities in isolation. Comparison with other helicases and mutational analyses show how it threads single-stranded RNA, and structural features suggest how it can load onto an internal region of U4/U6 di-snRNA. Although the C-terminal cassette does not seem to engage RNA in the same fashion, it binds ATP and strongly stimulates the N-terminal helicase. Mutations at the cassette interface, in an intercassette linker or in the C-terminal ATP pocket, affect this cross-talk in diverse ways. Together, our results reveal the structural and functional interplay between two helicase cassettes in a tandem superfamily 2 enzyme and point to several sites through which Brr2 activity may be regulated.

Common design principles in the spliceosomal RNA helicase Brr2 and in the Hel308 DNA helicase.

Brr2 is a unique DExD/H box protein required for catalytic activation and disassembly of the spliceosome. It contains two tandem helicase cassettes that both comprise dual RecA-like domains and a noncanonical Sec63 unit. The latter may bestow the enzyme with unique properties. We have determined crystal structures of the C-terminal Sec63 unit of yeast Brr2, revealing three domains, two of which resemble functional modules of a DNA helicase, Hel308, despite lacking significant sequence similarity. This structural similarity together with sequence conservation between the enzymes throughout the RecA-like domains and a winged helix domain allowed us to devise a structural model of the N-terminal active cassette of Brr2. We consolidated the model by rational mutagenesis combined with splicing and U4/U6 di-snRNA unwinding assays, highlighting how the RecA-like domains and the Sec63 unit form a functional entity that appears suitable for unidirectional and processive RNA duplex unwinding during spliceosome activation and disassembly.