Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase

Significance

RNA helicases can remodel substrate RNA–protein complexes in diverse ways. However, these activities have typically been studied using noncognate helicase-substrate systems, because the in vivo targets of many helicases are unknown. We investigated how the Brr2 protein disrupts its cognate U4/U6 di-small nuclear RNA–protein complex (di-snRNP), a reaction required for spliceosome catalytic activation. Our results suggest that Brr2 acts like a switch, initiating displacement of U6 snRNA, thereby inducing it to adopt an alternative conformation incompatible with base pairing to U4 snRNA. This mechanism explains how one U4/U6-bound protein, pre-mRNA processing factor 3 (Prp3), is displaced, whereas Prp31 and small nuclear ribonucleoprotein-associated protein 1 (Snu13) are released in complex with U4 snRNA. This unprecedented mode of RNP disruption minimizes requirements for reassembly of the U4/U6 di-snRNP after splicing.

Abstract

The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNA–protein complex (di-snRNP) during spliceosome activation via ATP-driven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as subcomplexes. Here, we set up a recombinant Brr2-mediated U4/U6 di-snRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein. Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31- and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing.