Ntr1 activates the Prp43 helicase to trigger release of lariat-intron from the spliceosome

Abstract

DEAD/H-box NTPases remodel the spliceosome at multiple steps during the pre-mRNA splicing cycle. The RNA-dependent NTPase Prp43 catalyzes dissociation of excised lariat-intron from the spliceosome, but it is unclear how Prp43 couples the energy of ATP hydrolysis to intron release. Here, we report that activation of Prp43's inherently feeble helicase activity by the splicing factor Ntr1 is required for lariat-intron release. Lethal Prp43 mutants T384A and T384V, which are active for ATP hydrolysis and fail to dissociate lariat-intron from spliceosomes, are refractory to stimulation of RNA unwinding by Ntr1. An N-terminal 120-amino-acid segment of Ntr1 suffices for binding to Prp43 and for stimulating its helicase activity. We identify missense mutations in Prp43 and Ntr1 that disrupt protein-protein interaction and impair Ntr1 enhancement of Prp43 RNA unwinding. Our results demonstrate for the first time that regulating the motor activity of a DEAH-box protein by an accessory factor is critical for mRNA splicing.[Keywords: Pre-mRNA splicing; DEAH-box helicase; Prp43; Ntr1; spliceosome] Supplemental material is available at http:/www.genesdev.org. Nuclear pre-mRNA splicing proceeds via two successive transesterification reactions (Burge et al. 1999). In the first step, the 2Ј-OH of the branch site adenosine within the intron attacks the phosphodiester at the 5Ј exonintron boundary to form a lariat-intermediate. In the second step, the 3Ј-OH of the 5Ј exon attacks the phosphodiester at the intron-3Ј exon junction to form mature mRNA and lariat-intron. Pre-mRNA splicing requires five snRNAs and >80 proteins (Will and Lührmann 2006). The small nuclear RNAs (snRNAs), packaged as snRNPs, assemble together with non-snRNP proteins onto the precursor RNA to form the spliceosome. The snRNPs are involved in identifying the splice junctions and positioning the reactive nucleotides for catalysis (Valadkhan 2005). ATP-dependent remodeling of RNA-RNA and RNA-protein interactions is essential for faithful spliceosome assembly, for the catalytic activation of the splice sites, and for spliceosome disassembly (Staley and Guthrie 1998).The remodeling events in the spliceosome are catalyzed by DExD/H NTPases that act at discrete stages of the splicing pathway (Staley and Guthrie 1998). Splicing factors Prp2, Prp16, Prp22, and Prp43 are members of the DEAH family of RNA-dependent NTPases (Burgess et al. 1990;Chen and Lin 1990;Company et al. 1991;Arenas and Abelson 1997). They are composed of a central NTPase/helicase domain, characteristic of all DExH proteins, plus an ∼300-amino-acid C-terminal domain unique to the Prp2/Prp16/Prp22/Prp43 clade (Tanner and Linder 2001;Silverman et al. 2003). The N-terminal segments are distinctive in each DEAH-box splicing factor. Understanding how the DEAH proteins carry out their specific functions during the splicing cycle requires knowledge about how they are recruited to the splicing complex, what their biochemical activities are, and whether they are regulated.Prp43 catalyzes the final step of ...

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

“…For example, Tsai et al (2007) have shown in a yeast two-hybrid assay that Ntr2 binds to Brr2, and, as Brr2 also binds to U5 snRNP and Prp43 binds to Ntr1, this would suggest a functional link among these components and the possibility that the formation of this complex would stimulate the helicase activity of Prp43 (Tanaka et al 2007). …”

“…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).…”

“…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). Ntr2 can also bind to Brr2, and it has been suggested that this interaction may help recruit Prp43 to the ILS (Tsai et al 2007).…”

Spliceosome discards intermediates via the DEAH box ATPase Prp43p

“…For example, the long branch site to 3′ splice site distance in the telomerase RNA intron may impede exon ligation by rendering the substrate sensitive to Prp22p-mediated rejection and Prp43p-mediated discard, thereby liberating the 3′ processed telomerase RNA. Finally, given the established role of Ntr1p/Spp382p in the activation of Prp43p (20), additional regulatory factors could target Prp43p to effectively modulate the partitioning of a substrate between discard and productive splicing and thereby to regulate this stage of RNA processing.…”

“…Whereas nuclear turnover may simply compete with splicing, cytoplasmic turnover implies that the spliceosome can dissociate a suboptimal substrate. After splicing, spliceosome disassembly and the dissociation of an optimal, excised intron require Ntr1p/Spp382p, Ntr2p, and the DEAH box ATPase Prp43p (20)(21)(22)(23)(24)(25), which also functions in the processing of pre-rRNA and histone pre-mRNA (26)(27)(28)(29). Interestingly, mutations in PRP43 and NTR1/SPP382 suppress mutations in the spliceosome assembly factors PRP38 as well as PRP8, and Ntr1p/Spp382p associates in vitro with stalled spliceosomes that retain the lariat intermediate but lack the 5′ exon, hinting that the spliceosome discards intermediates by a mechanism that parallels the mechanism for discarding an optimal, excised intron product (30).…”