Yeast ntr1/spp382 mediates prp43 function in postspliceosomes

Abstract

The Ntr1 and Ntr2 proteins of Saccharomyces cerevisiae have been reported to interact with proteins involved in pre-mRNA splicing, but their roles in the splicing process are unknown. We show here that they associate with a postsplicing complex containing the excised intron and the spliceosomal U2, U5, and U6 snRNAs, supporting a link with a late stage in the pre-mRNA splicing process. Extract from cells that had been metabolically depleted of Ntr1 has low splicing activity and accumulates the excised intron. Also, the level of U4/U6 di-snRNP is increased but those of the free U5 and U6 snRNPs are decreased in Ntr1-depleted extract, and increased levels of U2 and decreased levels of U4 are found associated with the U5 snRNP protein Prp8. These results suggest a requirement for Ntr1 for turnover of the excised intron complex and recycling of snRNPs. Ntr1 interacts directly or indirectly with the intron release factor Prp43 and is required for its association with the excised intron. We propose that Ntr1 promotes release of excised introns from splicing complexes by acting as a spliceosome receptor or RNA-targeting factor for Prp43, possibly assisted by the Ntr2 protein.

FIG. 1.

Ntr1 and Ntr2 associate with snRNPs and excised intron RNA. (A) Coprecipitation of snRNAs with Ntr1 and Ntr2. Splicing extracts (50 μl) with nontagged proteins (BMA38a; lane 4), Ntr2-TAP (KL2T; lane 5), or Ntr1-TAP (KL4T; lane 6) were mixed with an equal volume of precipitation buffer containing IgG-agarose beads and incubated at 4°C for 2 h. The beads were washed in buffer containing 150 mM NaCl and deproteinized, and the RNAs were precipitated, fractionated in an 8% polyacrylamide-8 M urea gel, blotted, and probed for U1, U2, U4, U5, and U6 snRNAs. Total RNA extracted from 12.5 μl of each extract is shown in lanes 1 to 3. (B) Coprecipitation of splicing complexes by Ntr1 and Ntr2. Splicing extracts derived from cultures with nontagged proteins (BMA38a; lanes 1 and 6), Prp8-TAP (RG8T; lanes 2 and 7), Ntr1-TAP (KL4T; lanes 3 and 8), Ntr2-TAP (KL2T; lanes 4 and 9), and Ntr2-TAP with HA-Ntr1 depleted (KL4G2T cells grown in glucose for 10 h; lanes 5 and 10) were incubated under splicing conditions in vitro (60-μl total volume) with ³²P-labeled ACT1 RNA as the substrate. The reactions were stopped after 25 min, and 10-μl volumes were removed as splicing controls (not shown). The remaining 50-μl volumes were mixed with IgG-agarose beads and incubated at 4°C for 2 h. Beads were washed in buffer containing IPP150 and deproteinized, and the RNAs were precipitated. The unbound samples were also deproteinized, and 20% aliquots were analyzed (lanes 1 to 5) alongside the precipitates (lanes 6 to 10) by fractionation in a 7% polyacrylamide-8 M urea gel and autoradiography. The various RNA species are indicated diagrammatically to the right of panel B, with rectangles representing exons, a thin line representing the intron, and a lariat loop representing the branched form of the intron following the first catalytic step of splicing. IP, immunoprecipitation.

FIG. 2.

Effects of Ntr1 depletion on splicing in vitro. (A) Northern analysis of U3 snoRNA. BMA38 (lanes 1 to 5) or KL4G (P_GAL1-3HA-NTR1; lanes 6 to 10) cells were grown in YPGR (galactose medium) to an OD₆₀₀ of 0.5 and then transferred to YPD (glucose medium; repressing conditions). RNA was extracted after 0, 2, 4, 6, or 10 h following the shift, subjected to denaturing polyacrylamide gel electrophoresis, blotted, and probed for U3A snoRNA. (B and C) KL4G cells were grown in YPGR to an OD₆₀₀ of 0.5 and then transferred to YPD and grown for 10 h before harvesting to produce a splicing extract. A nondepleted control culture of KL4G was produced by similar treatment but with a return to YPGR instead of YPD. (B) Western blot assay showing the relative amounts of HA-tagged Ntr1 in KL4G cells at 0 h (lane 1) and at 10 h (lane 2) after the shift to YPD. Nop1 was probed as a loading control. (C) In vitro splicing assays with extracts derived from Ntr1-depleted (lanes 4 to 6) and nondepleted (lanes 1 to 3) cells. Aliquots (5 μl) were withdrawn and halted after incubation for 0, 10, and 25 min at 23°C. The RNAs were analyzed by fractionation in a 7% polyacrylamide-8 M urea gel and autoradiography. Splicing reaction mixtures with Ntr1-depleted (lanes 9 and 11) and nondepleted (lanes 7, 8, and 10) extracts were incubated with preimmune (PI) serum (lane 7), anti-Prp8 antibodies (lane 8 and 9), or anti-Cef1 antibodies (lanes 10 and 11), and precipitated RNAs were extracted and analyzed as described above. The various RNA species are indicated diagrammatically on the right as described in the legend to Fig. 1. IP, immunoprecipitation.

FIG. 3.

Depletion of Ntr1 leads to a redistribution of snRNP complexes. To examine the profile of snRNP distribution, non-Ntr1-depleted (A) and Ntr1-depleted (B) cell extracts (no added ATP or pre-mRNA) were centrifuged through 10 to 30% glycerol gradients and RNAs were extracted from alternate fractions and analyzed by Northern blotting with hybridization with probes for U1, U2, U4, U5, and U6 snRNAs. The sedimentation of snRNP complexes is indicated. In addition to U4/U6.U5 tri-snRNPs and U5 snRNPs, fractions 1 to 7 may contain some endogenous spliceosome and postsplicing complexes. (C) Coprecipitation of snRNAs by anti-Prp8 antibodies in extracts from Ntr1-depleted (lanes 4 to 6) or nondepleted (lanes 1 to 3) cells. RNAs were analyzed by fractionation in a 6% polyacrylamide-8 M urea gel and Northern blotting. Preimmune (PI) serum was used as a background control. (D) Same as panel C, but the precipitated snRNAs were analyzed by nondenaturing gel electrophoresis as described previously (41). IP, immunoprecipitation.

FIG. 4.

Associations among Ntr1, Ntr2, Prp43, and Prp8. (A and B) Glycerol gradient fractionation (without added ATP or pre-mRNA). (A) Extract from galactose-grown KL4G2T cells containing HA-Ntr1 and Ntr2-TAP was fractionated in a 10 to 30% glycerol gradient. Alternate gradient fractions were immunoprecipitated with IgG-agarose, and the precipitated proteins were analyzed by Western blotting with anti-HA and anti-Prp8 (8.6) antibodies. (B) KL4G cell extract containing HA-Ntr1 was fractionated in a 10 to 30% glycerol gradient. Alternate gradient fractions were immunoprecipitated with anti-HA agarose, and the precipitated proteins were analyzed by Western blotting with affinity-purified anti-Prp43 antibodies and with anti-Prp8 antibodies. (C) Extracts (without added ATP or pre-mRNA) from galactose-grown (lanes 2 and 5) or glucose-grown (lanes 3 and 6) KL4G2T cells or from BMA38a (untagged control) cells (lanes 1 and 4) were incubated with IgG-agarose to precipitate Ntr2-TAP, and the precipitated proteins were analyzed by Western blotting with anti-HA and anti-Prp8 antibodies. Note that the Ntr2-TAP protein in whole extract is very poorly recognized by IgG on the blot. IP, immunoprecipitation.

FIG. 5.

Immunoprecipitation (IP) of splicing reaction mixtures shows that Ntr1 is required for association of Prp43 with the excised intron. Ntr1-depleted (lanes 2, 3, 5, and 6) and nondepleted (lanes 1 and 4) extracts were prepared from KL4G cells grown as described in the legend to Fig. 2. Splicing reaction mixtures (50 μl) were assembled with ³²P-labeled ACT1 substrate RNA and 0.9 μg of purified recombinant Prp43_T123A. For reconstitution of the Ntr1-depleted extract (lanes 3 and 6), the splicing reaction mixture was mixed with 3HA-Ntr1 affinity selected from galactose-grown KL4G cell extract (overproducing 3HA-Ntr1). Following incubation for 25 min at room temperature, 90% of each splicing reaction mixture was mixed with 20 μl of affinity-purified anti-Prp43 antibodies immobilized on Sepharose CL45 beads in IPP150. After mixing at 4°C for 1 h, the beads were washed and the RNA was extracted. Precipitated RNA (lanes 4 to 6) and RNA extracted from 10% aliquots of the total reaction mixtures (lanes 1 to 3) were separated on a 7% polyacrylamide-8 M urea gel. For each reconstitution reaction mixture, 10 μl of extract from YPGR-grown KL4G cells was incubated with anti-HA-agarose beads for 1 h at 4°C and the beads were washed five times with IPP150 buffer and once in 0.6 M potassium phosphate buffer (pH 7) and then incubated with 0.5 μg of HA peptide (Sigma) for 8 h at 4°C to elute the HA-Ntr1 from the beads. Recombinant His₁₀-tagged Prp43_T123A protein was produced in Escherichia coli BL21-CodonPlus(DE3)RIL (Stratagene) and purified essentially as described by Martin et al. (26). The various RNA species are indicated diagrammatically on the right as described in the legend to Fig. 1.