Spliceosome disassembly catalyzed by Prp43 and its associated components Ntr1 and Ntr2

Abstract

Two novel yeast splicing factors required for spliceosome disassembly have been identified. Ntr1 and Ntr2 (NineTeen complex-Related proteins) were identified for their weak association with components of the Prp19-associated complex. Unlike other Prp19-associated components, these two proteins were primarily associated with the intron-containing spliceosome during the splicing reaction. Extracts depleted of Ntr1 or Ntr2 exhibited full splicing activity, but accumulated large amounts of lariat-intron in the spliceosome after splicing, indicating that the normal function of the Prp19-associated complex in spliceosome activation was not affected, but spliceosome disassembly was hindered. Immunoprecipitation analysis revealed that Ntr1 and Ntr2 formed a stable complex with DExD/H-box RNA helicase Prp43 in the splicing extract. Ntr1 interacted with Prp43 through the N-terminal G-patch domain, with Ntr2 through a middle region, and with itself through the carboxyl half of the protein. The affinity-purified Ntr1-Ntr2-Prp43 complex could catalyze disassembly of the spliceosome in an ATP-dependent manner, separating U2, U5, U6, NTC (NineTeen Complex), and lariat-intron. This is the first demonstration of physical disassembly of the spliceosome, catalyzed by a complex containing a DExD/H-box RNA helicase and two accessory factors, which might function in targeting the helicase to the correct substrate.[Keywords: Spliceosome disassembly; NTR complex; Prp43; Ntr1; Ntr2] Supplemental material is available at http://www.genesdev.org.

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

“…Our results are thus consistent with the idea that an intact U2 snRNP is released from the ILS in vivo as well. It was reported previously that the U2 snRNP dissociated from the ILS by the NTR complex is likely missing some proteins or has an altered conformation (Tsai et al 2005). The different association behavior of the U2 proteins observed here is probably due to differences in the stringencies of the experimental conditions used in these two studies.…”

“…At the same time, the remaining spliceosomal core, containing the U2/U6 and U5 snRNPs, is also disassembled (Tsai et al 2005). Yeast Prp43 interacts with its cofactors, Ntr1 (also called Spp382) and Ntr2, forming the NTR complex (Tsai et al 2005;Boon et al 2006).…”

“…At the same time, the remaining spliceosomal core, containing the U2/U6 and U5 snRNPs, is also disassembled (Tsai et al 2005). Yeast Prp43 interacts with its cofactors, Ntr1 (also called Spp382) and Ntr2, forming the NTR complex (Tsai et al 2005;Boon et al 2006). Ntr2 forms a tight complex with Ntr1, while Ntr1 interacts with Prp43 through its G-patch domain (Tsai et al 2005;Tanaka et al 2007).…”

“…The dissociation of the IL RNA from the ILS requires the DEAH-box RNA helicase Prp43 (Arenas and Abelson 1997;Martin et al 2002;Tsai et al 2005). At the same time, the remaining spliceosomal core, containing the U2/U6 and U5 snRNPs, is also disassembled (Tsai et al 2005).…”

“…Yeast Prp43 interacts with its cofactors, Ntr1 (also called Spp382) and Ntr2, forming the NTR complex (Tsai et al 2005;Boon et al 2006). Ntr2 forms a tight complex with Ntr1, while Ntr1 interacts with Prp43 through its G-patch domain (Tsai et al 2005;Tanaka et al 2007). The latter interaction is crucial for the stimulation of Prp43's otherwise weak RNA helicase activity, which is essential for IL release (Tanaka et al 2007).…”