Sad1 counteracts Brr2-mediated dissociation of U4/U6.U5 in tri-snRNP homeostasis.Mol

The yeast Sad1 protein was previously identified in a screen for factors involved in the assembly of the U4/U6 di-snRNP particle. Sad1 is required for pre-mRNA splicing both in vivo and in vitro, and its human orthologue has been shown to associate with U4/U6.U5 tri-snRNP.We show here that Sad1 plays a role in maintaining a functional form of the tri-snRNP by promoting the association of U5 snRNP with U4/U6 di-snRNP. In the absence of Sad1, the U4/U6.U5 tri-snRNP dissociates into U5 and U4/U6 upon ATP hydrolysis and cannot bind to the spliceosome. The separated U4/U6 and U5 can reassociate upon incubationmore favorably in the absence of ATP and in the presence of Sad1. Brr2 is responsible for mediating ATP-dependent dissociation of the tri-snRNP. Our results demonstrate a role of Sad1 in maintaining the integrity of the tri-snRNP by counteracting Brr2-medi-ated dissociation of tri-snRNP and provide insights into homeostasis of the tri-snRNP. Splicing of precursor mRNA takes place via two steps of trans-esterification reactions and is catalyzed by a large ribonucleo-protein complex called the spliceosome. The spliceosome consists of five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6, and nearly a hundred proteins. These snRNAs are associated with specific protein components to form small nuclear ribonucleo-protein particles (snRNPs), among which U4 and U6 base pair with each other to form a U4/U6 di-snRNP, which can furthe