Rds3p is required for stable U2 snRNP recruitment to the splicing apparatus - PubMed (original) (raw)
Rds3p is required for stable U2 snRNP recruitment to the splicing apparatus
Qiang Wang et al. Mol Cell Biol. 2003 Oct.
Abstract
Rds3p is a well-conserved 12-kDa protein with five CxxC zinc fingers that has been implicated in the activation of certain drug transport genes and in the pre-mRNA splicing pathway. Here we show that Rds3p resides in the yeast spliceosome and is essential for splicing in vitro. Rds3p purified from yeast stably associates with at least five U2 snRNP proteins, Cus1p, Hsh49p, Hsh155p, Rse1p, and Ist3p/Snu17p, and with the Yra1p RNA export factor. A mutation upstream of the first Rds3p zinc finger causes the conditional release of the putative branchpoint nucleotide binding protein, Ist3p/Snu17p, and weakens Rse1p interaction with the Rds3p complex. The resultant U2 snRNP particle migrates exceptionally slowly in polyacrylamide gels, suggestive of a disorganized structure. U2 snRNPs depleted of Rds3p fail to form stable prespliceosomes, although U2 snRNA stability is not affected. Metabolic depletion of Yra1p blocks cell growth but not splicing, suggesting that Yra1p association with Rds3p relates to Yra1p's role in RNA trafficking. Together these data establish Rds3p as an essential component of the U2 snRNP SF3b complex and suggest a new link between the nuclear processes of pre-mRNA splicing and RNA export.
Figures
FIG. 1.
Rds3p activity is required for pre-mRNA splicing. (A) Northern blot analysis of RNA isolated from yeast strains that express a wild-type RDS3 gene and from yeast strains that express a glucose-repressible GAL1::rds3-1 allele. Samples were taken from yeast grown constitutively on galactose at room temperature (Gal, 23°C) and after shift to glucose-based medium (Glu) for 10 h at 37°C. (B) In vitro assay of splicing conducted in the presence and absence of active Rds3p or Rds3-TAP. The extract used in lanes 4 to 9 and 12 to 18 was preincubated with the TAP-specific calmodulin and IgG affinity resins prior to the assay. In lanes 22 to 27, extract was prepared from the glucose-repressed, heat-inactivated GAL1::rds3-1 mutant. For lanes 7 to 9, 16 to 18, and 25 to 28 the double-affinity-purified Rds3-TAP complex was included in the splicing reaction mixture. The positions of the RPS17A pre-mRNA (P), lariat intermediate (LI), 5′ exon (5′E), mRNA (M), and excised intron (I) are indicated.
FIG. 2.
Rds3p is present in the yeast spliceosome. (A) Western blot of whole-cell yeast extracts (lanes 1 to 3) and affinity-purified splicing complexes (lanes 4 to 7) probed with a TAP-specific antibody. Spliceosomes were assembled for 45 min on biotin-substituted (+) or unmodified (−) RPS17A pre-mRNA in wild-type extracts (Rds3p) and in extracts where the epitope-tagged Rds3-TAP or Clf1-TAP served as the only source of these essential proteins. The positions of the relevant tagged proteins are indicated on the right. Strept, streptavidin.
FIG. 3.
Rds3p is required for the commitment complex to prespliceosome transition. (A) Native gel electrophoresis of splicing complexes assembled on RPS17A pre-mRNA. Rds3-TAP splicing extract was assayed without additional manipulation (lanes 1 to 3) and after preabsorption with calmodulin agarose and protein A agarose affinity resins (lanes 4 to 6). Extracts were also prepared from glucose-grown, temperature-inactivated GAL1::rds3-1 yeast (lanes 7 to 9 and 13 to 15) and from wild-type yeast (lanes 10 to 12). The wild-type extract was preincubated with RNase H and an anti-U2 oligonucleotide prior to assay in order to block assembly at the commitment complex stage. In lanes 13 to 15, the extract was preincubated with pre-mRNA for 5 min prior to the addition of a 50-fold excess of unlabeled substrate and the complementing Rds3-TAP complex. The positions of the unassembled pre-mRNA (U), commitment complex bands (CC1 and CC2), prespliceosome (III), snRNP complete splicing complex (I), and catalytically active spliceosome (II) are indicated at the left. (B) Northern analysis of splicing complex snRNAs. Shown is a membrane transfer of RNA present in wild-type extract (Rds3p) and extract prepared from the glucose-grown, temperature-inactivated GAL1::rds3-1 culture (Rds3-1p dep.) probed for the spliceosomal snRNAs. RNA in the unfractionated extract (Total) is compared with that released from affinity-purified splicing complexes assembled on biotin-substituted or unmodified (no biotin) pre-mRNA. The positions of the spliceosomal snRNAs are noted at the left.
FIG. 4.
Protein characterization of the Rds3-TAP complex. (A) Profile of Rds3-TAP-associated proteins. 35S-labeled proteins from Rds3-TAP or the wild-type control (Rds3p) cultures were isolated by the two-step TAP affinity purification and glycerol gradient separation (see Materials and Methods) and resolved on a sodium dodecyl sulfate-5 to 10% gradient polyacrylamide gel with Benchmark molecular mass markers (indicated on right in kilodaltons). Bands present in the Rds3p control are background proteins. The protein assignments in the Rds3-TAP lane were made based on mass analysis of tryptic digests of gel bands and of the entire complex without prior gel fractionation (see Materials and Methods). (B) Ist3p/Snu17p is present in the Rds3p complex. The bands correspond to proteins recovered by Rds3p-TAP affinity purification in a wild-type (IST3/SNU17) and null allele (ist3::Kanr) background. The background bands are somewhat greater here than in panel A since the Rds3-TAP complex was not gradient fractionated prior to analysis. A minor background band migrates with or just above the position of Ist3p/Snu17p. (C) The prominent Rds3p complex bands are RNase resistant. Rds3-TAP yeast extract was used for affinity purification (without gradient separation) before and after extensive digestion with RNase A and RNase T1. The first lane (Rds3p) shows the positions of background proteins. (D) The Rds3p complex is salt stable. Rds3-TAP extracts were adjusted to the indicated NaCl level during the binding and wash steps of protein A-IgG selection. Subsequent calmodulin agarose chromatography was conducted under standard (150 mM) salt conditions (62).
FIG. 5.
Rds3-1p depletion specifically alters the U2 snRNP particle. Yeast snRNP particles prepared from wild-type yeast (Rds3p) and GAL1::rds3-1 yeast (Rds3-1p dep.) were resolved by native gel electrophoresis and hybridized with snRNA-specific probes. Both cultures were grown in glucose-based medium at 30°C and shifted to 37°C for 1 h prior to extract preparation. The positions of the various snRNP complexes resolved are indicated.
FIG. 6.
Rds3-1-TAP inactivation impairs Ist3p/Snu17p and Rse1p association with the Rds3p complex. Radiolabeled proteins were recovered by TAP purification (without gradient separation) from wild-type (Rds3-TAP) and mutant (Rds3-1-TAP) cultures grown constitutively at 30°C or shifted to 37°C for 1 h prior to harvest. The asterisk indicates the position of reproducible bands lost or greatly reduced with temperature shift.
FIG. 7.
Metabolic depletion of Yra1p does not impair pre-mRNA splicing. RNA was isolated from the GAL1::_YRA1_-dependent yeast culture grown constitutively in galactose (Gal) and after shift to glucose medium for the indicated periods of time. The RNA was fractionated on a denaturing formaldehyde gel and hybridized with probes for the intron-bearing RPS17A (formerly known as RP51A) transcripts, the intronless ADE3 mRNA, and the U2 snRNA.
FIG. 8.
Not all Yra1p is associated with Rds3p. A Western blot of extracts prepared from yeast that express Yra1-ProtA only or Yra1-ProtA and Rds3-TAP simultaneously was probed with antibodies to the common protein A component. Total, unfractionated extract; Bound, proteins released from calmodulin agarose. No protein A agarose was used as this would select for both tagged proteins. Approximately 10 extract equivalents were loaded in the Bound lane compared to the Total lane.
FIG. 9.
Model for Rds3p function and Yra1p recruitment in splicing. Assembly of the splicing apparatus from the branchpoint-dependent commitment complex (CC2) through the point of mRNA release is diagrammed. For clarity's sake only a subset of the splicing-related snRNP and non-snRNP proteins are indicated. Rds3p is shown as essential for the stable addition of the U2 snRNP particle to the commitment complex in prespliceosome formation. Yra1p is presented as being either associated with the U2 snRNP or recruited independently to the spliceosome through contacts that might include both Sub2p and one or more components of the Rds3p-SF3b complex. Sometime prior to or concurrent with the Prp22p-dependent mRNA release step, components of the RNA export pathway are deposited on the processed mRNA. Rds3p likely remains stably associated with the snRNP components of the splicing apparatus, although its release with the RNA export factors cannot be ruled out.
References
- Aebersold, R., and M. Mann. 2003. Mass spectrometry-based proteomics. Nature 422:198-207. - PubMed
- Akache, B., and B. Turcotte. 2002. New regulators of drug sensitivity in the family of yeast zinc cluster proteins. J. Biol. Chem. 277:21254-21260. - PubMed
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