title: A role for ubiquitin in the spliceosome assembly pathway.
abstract: The spliceosome uses numerous strategies to regulate its function in mRNA maturation. Ubiquitin regulates many cellular processes, but its potential roles during splicing are unknown. We have developed a new strategy that reveals a direct role for ubiquitin in the dynamics of splicing complexes. A ubiquitin mutant (I44A) that can enter the conjugation pathway but is compromised in downstream functions diminishes splicing activity by reducing the levels of the U4/U6-U5 small nuclear ribonucleoprotein (snRNP). Similarly, an inhibitor of ubiquitin's protein-protein interactions, ubistatin A, reduces U4/U6-U5 triple snRNP levels in vitro. When ubiquitin interactions are blocked, ATP-dependent disassembly of purified U4/U6-U5 particles is accelerated, indicating a direct role for ubiquitin in repressing U4/U6 unwinding. Finally, we show that the conserved splicing factor Prp8 is ubiquitinated within purified triple snRNPs. These results reveal a previously unknown ubiquitin-dependent mechanism for controlling the pre-mRNA splicing pathway.
authors.affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, Illinois 60208, USA.
authors.forename: Priya
authors.initials: P
authors.lastname: Bellare
authors.forename: Eliza C
authors.initials: EC
authors.lastname: Small
authors.forename: Xinhua
authors.initials: X
authors.lastname: Huang
authors.forename: James A
authors.initials: JA
authors.lastname: Wohlschlegel
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Staley
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Sontheimer
mesh_terms: D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D011804:Quinolines / Q000494:pharmacology; D012326:RNA Splicing; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*; D013425:Sulfanilic Acids / Q000494:pharmacology; D025801:Ubiquitin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: C077018:PRP8 protein, S cerevisiae; D011804:Quinolines; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D013425:Sulfanilic Acids; D025801:Ubiquitin; C492595:ubistatin A