Inhibition of a spliceosome turnover pathway suppresses splicing defects

Abstract

Defects in assembly are suggested to signal the dissociation of faulty splicing complexes. A yeast genetic screen was performed to identify components of the putative discard pathway. Weak mutant alleles of SPP382 (also called NTR1) were found to suppress defects in two proteins required for spliceosome activation, Prp38p and Prp8p. Spp382p is shown necessary for cellular splicing, with premRNA and, for some alleles, excised intron, accumulating after inactivation. Like spp382-1, a mutant allele of AAR2 was identified in this suppressor screen. Like Spp382p, Aar2p has a reported role in spliceosome recycling and is found with Spp382p in a complex recovered with a mutant version of the spliceosomal core protein Prp8p. Possible insight into to the spp382 suppressor phenotype is provided by the observation that defective splicing complexes lacking the 5' exon cleavage intermediate are recovered by a tandem affinity purification-tagged Spp382 derivative. Stringent proteomic and two-hybrid analyses show that Spp382p also interacts with Cwc23p, a DNA J-like protein present in the spliceosome and copurified with the Prp43p DExD/H-box ATPase. Spp382p binds Prp43p and Prp43p requires Spp382p for intron release from the spliceosome. Consistent with a related function in the removal of defective complexes, three prp43 mutants are also shown to suppress splicing defects, with efficiencies inversely proportionate to the measured ATPase activities. These and related genetic data support the existence of a Spp382p-dependent turnover pathway acting on defective spliceosomes.

Fig. 1.

The recessive spp382-1 mutation suppresses prp38-1. (A) Growth of wild-type yeast (streaks 1 and 5), an otherwise isogenic prp38-1 (ts192) mutant (streaks 2 and 6), and the suppressed mutant (streaks 3 and 4) on rich media after 3 days at the permissive temperature (23°C) or 2 days at the restrictive temperature (37°C). In streaks 4–6, a centromeric yeast plasmid with the wild-type SPP382 gene (pSPP382) is coexpressed. (B) Growth of wild-type yeast (PRP38 and AAR2), the prp38-1 mutant, and the suppressed prp38-1, aar2-D281N double mutant after 2 days at 37°C. (C) PCR-based mutagenesis of conserved regions of Spp382p (based on UniProtKB entries: Q06411, Q9UBB9, Q6DI35, Q9NHN7, and Q17784). The amino acid changes and numbered coordinates on the 708-aa protein are indicated above the bar.

Fig. 2.

Loss of Spp382p activity impairs splicing. (A) Northern analysis of splicing inhibition in yeast that express the nutritionally regulated GAL1::spp382-4 gene (T = 0) and 4–20 h after transcriptional repression by glucose. Wild-type yeast (SPP382) were assayed in parallel grown in galactose (T = 0) or glucose (T− = 20) media. The positions of the intronless ADE3 mRNA and the RPS17A premRNA and mRNA are shown to the left. (B Upper) Northern blot analysis as in A with wild-type yeast, the prp38-1 mutant, and the nonlethal spp382 mutants grown at room temperature (−) or after 2 h at 37°C (+). (B Lower) Accumulation of the ACT1 excised intron RNA. (C) Splicing of the prp38-1 mutant and the prp38-1, spp382-1 double mutant at 23°C and after 2 h at 37°C (+). The 25S and 18S rRNA bands are presented as normalization controls for RNA loading and transfer.

Fig. 3.

Spp382p binds defective spliceosomes. (A) In vitro premRNA splicing with ³²P-labeled RPS17A premRNA processed for the indicated times under standard conditions and assayed by denaturing PAGE before (Total) or after (IgG) IgG agarose selection. The extracts were prepared from the indicated TAP-tagged strains and from an untagged control (WT). The detail (det.) to the right shows a 4-fold overexposure of the first six lanes to highlight the absence of upstream exon with IgG-agarose selected Spp382-TAP. The positions of the unprocessed premRNA (P), lariat intermediate (LI), excised intron (I), spliced mRNA (M), and free upstream exon (5′E) are indicated. (B) In vitro splicing and RNA recovery with a 3′ splice site truncated RPS17A mRNA. Alternating lanes show the total processed RNA (T) and the IgG agarose pellets (P) after 30 min of splicing. Lanes 5–8 show the results of splicing reactions in which the BBP-TAP extract was preincubated with endogenous RNase H and an oligonucleotide against the U2 snRNA (α U2) or a nonspecific control oligonucleotide (cont.). The asterisk shows the position of linearized lariat intermediate sometimes observed under these conditions. The detail below (det.) shows a 4-fold underexposure of the corresponding premRNA region to highlight the enhanced substrate recovery by IgG agarose after U2 snRNA degradation. (C) Extended (+13) or shortened (Δ16, Δ25) 5′ exon RPS17A substrates were spliced for 30 min under standard conditions and then assayed for products before (lanes 1–4) or after (lanes 5–8) Spp382-TAP selection. The 5′ exons (below the bar) were run on a second gel to resolve the smaller fragments. In all panels, ≈20-fold more IgG sample is loaded relative to the unfractionated (total) RNA sample. The RNA band intensities were determined with ImageQuant 5.2 (GE Healthcare, Piscataway, NJ).

Fig. 4.

Genetic characterization of extragenic suppression. (A) Yeast with ts prp38-1, prp8-1 or prp4-1 mutations were transformed with a plasmid to overexpress the prp38-1 mutant (GAL1::prp38-1) or the previously characterized SPP381 dosage suppressor (GAL1::SPP381). GAL1::spp382-4 was expressed in a prp38-1, SPP382::Kan^R background. The cultures were serially diluted and separately plated on yeast extract/peptone/dextrose medium at the restrictive temperature for prp8-1 (30°C) and prp38-1 (37°C). To detect even minor changes in growth, a semipermissive temperature of 30°C was used for prp4-1. (B) Three mutant alleles of PRP43 assayed for growth at 36°C in the prp38-1 or wild-type (PRP38) background. The Prp43p ATPase activities are from Martin et al. (39).