Sad1 Counteracts Brr2-Mediated Dissociation of U4/U6.U5 in Tri-snRNP Homeostasis

Abstract

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 incubation more 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-mediated dissociation of tri-snRNP and provide insights into homeostasis of the tri-snRNP.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00837-13.

ACKNOWLEDGMENTS

We thank A. Peña for English editing and members of the Cheng laboratory for helpful discussions.

This work was supported by a grant from the Academia Sinica and the National Science Council (Taiwan) (NSC101-2745-B-001-001-ASP).