Brr2p-mediated unwinding of U4/U6 is promoted by a mutually exclusive internal stem loop in U4 and involves destabilization of the 5’ stem-loop of U4

Before the spliceosome engages a pre-mRNA to excise its introns, the catalytic small nuclear RNA (snRNA) U6 is inactive because of base pairing with U4 snRNA; thus, spliceosome activation requires unwinding of base paired U4/U6, composed of stem I and stem II. The Ski2-like ATPase and RNA helicase Brr2p facilitates U4/U6 unwinding and the ultimately irreversible release of U4; however, the molecular mechanism behind Brr2p-mediated U4/U6 unwinding and the roles of the snRNAs in unwinding remain incompletely understood. To investigate the mechanism in vivo in budding yeast, we screened an unwinding-deficient, cold-sensitive brr2 mutant, associated with retinitis pigmentosa in humans, for genetic interactions with mutations in U4 snRNA. Some destabilizing U4 mutations in either stem I or stem II suppressed the brr2 mutant, providing functional evidence that Brr2p disrupts both stems in vivo. Further, destabilizing mutations in the intervening 5’ stem loop of U4 also suppressed the brr2 mutant, and in vitro Brr2p displaced Prp31p from this stem loop, implicating Brr2p in disruption of this structure, too. Unexpectedly and counterintuitively, a subset of destabilizing mutations in U4/U6 stem I exacerbated the brr2 mutant. These mutations disrupted a conserved, internal stem loop in U4 that is mutually exclusive with U4/U6 stem I. In yeast, we found that this stem loop (U4-ISL1) is required for splicing in vivo and for U4/U6 unwinding in vitro. Notably, this stem loop overlaps with a region of human RNU4-2 mutated in neurodevelopmental disorders. Altogether, these results implicate Brr2p in disrupting all U4 secondary structures upstream of its initial U4 binding site and implicate an important role for U4 in antagonizing U4/U6 reannealing during Brr2p-mediated U4/U6 unwinding.