A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles

Science

Volumn 279, Issue 5352, 1998, Pages 857-860

  Raghunathan, Pratima L ^a   Guthrie, Christine ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ADENOSINE TRIPHOSPHATE; GLUCOSE; MESSENGER RNA PRECURSOR; RNA BINDING PROTEIN; SMALL NUCLEAR RIBONUCLEOPROTEIN; SMALL NUCLEAR RNA;

ARTICLE; CATALYSIS; CONTROLLED STUDY; ESCHERICHIA COLI; GEL ELECTROPHORESIS; NONHUMAN; PRIORITY JOURNAL; RNA SPLICING; SPLICEOSOME; YEAST;

ADENOSINE TRIPHOSPHATE; BASE COMPOSITION; FUNGAL PROTEINS; MODELS, GENETIC; RIBONUCLEOPROTEIN, U4-U6 SMALL NUCLEAR; RNA SPLICING; RNA, FUNGAL; RNA, SMALL NUCLEAR; RNA-BINDING PROTEINS; SACCHAROMYCES CEREVISIAE; SPLICEOSOMES;

ESCHERICHIA COLI;

EID: 0032488860     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5352.857     Document Type: Article

References (51)

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18. A Not I site was introduced immediately upstream of the stop codon of the PRP24 gene by polymerase chain reaction (PCR); no mutations were found in the amplified region. The 121-base pair (bp) triple hemagglutinin (3HA) tag [M. Tyers and B. Futcher, Mol. Cell. Biol. 13, 5659 (1993)] and 54-bp polyoma (Pya) tag (32) were separately inserted into the Not I site, and the resulting PRP24-3HA and PRP24-Pya fragments were subcloned into pUN90 [S. J. Elledge and R. W. Davis, Gene 70, 303]. These plasmids were transformed into PRY98 (MATα his3Δ trp1Δ lys2-801 ura3-52 ade2-101 PRP24::LYS2 pUN50-PRP24) and were shown to complement the PRP24::LYS2 deletion by plasmid shuffling on 5-fluoroorotic acid [C. Guthrie and G. R. Fink, Eds., Guide to Yeast Genetics and Molecular Biology (Academic Press, San Diego, CA, 1991)]. The resulting PRP24-3HA strain (PRY112, or PRY98 with pUN90-PRP24-3HA) was used for biochemical analysis.
19. A Not I site was introduced immediately upstream of the stop codon of the PRP24 gene by polymerase chain reaction (PCR); no mutations were found in the amplified region. The 121-base pair (bp) triple hemagglutinin (3HA) tag [M. Tyers and B. Futcher, Mol. Cell. Biol. 13, 5659 (1993)] and 54-bp polyoma (Pya) tag (32) were separately inserted into the Not I site, and the resulting PRP24-3HA and PRP24-Pya fragments were subcloned into pUN90 [S. J. Elledge and R. W. Davis, Gene 70, 303]. These plasmids were transformed into PRY98 (MATα his3Δ trp1Δ lys2-801 ura3-52 ade2-101 PRP24::LYS2 pUN50-PRP24) and were shown to complement the PRP24::LYS2 deletion by plasmid shuffling on 5-fluoroorotic acid [C. Guthrie and G. R. Fink, Eds., Guide to Yeast Genetics and Molecular Biology (Academic Press, San Diego, CA, 1991)]. The resulting PRP24-3HA strain (PRY112, or PRY98 with pUN90-PRP24-3HA) was used for biochemical analysis.
20. A Not I site was introduced immediately upstream of the stop codon of the PRP24 gene by polymerase chain reaction (PCR); no mutations were found in the amplified region. The 121-base pair (bp) triple hemagglutinin (3HA) tag [M. Tyers and B. Futcher, Mol. Cell. Biol. 13, 5659 (1993)] and 54-bp polyoma (Pya) tag (32) were separately inserted into the Not I site, and the resulting PRP24-3HA and PRP24-Pya fragments were subcloned into pUN90 [S. J. Elledge and R. W. Davis, Gene 70, 303]. These plasmids were transformed into PRY98 (MATα his3Δ trp1Δ lys2-801 ura3-52 ade2-101 PRP24::LYS2 pUN50-PRP24) and were shown to complement the PRP24::LYS2 deletion by plasmid shuffling on 5-fluoroorotic acid [C. Guthrie and G. R. Fink, Eds., Guide to Yeast Genetics and Molecular Biology (Academic Press, San Diego, CA, 1991)]. The resulting PRP24-3HA strain (PRY112, or PRY98 with pUN90-PRP24-3HA) was used for biochemical analysis.
21. Aliquots (100 μl) of Prp24-3HA extract were incubated with 5 μg of 12CA5 antibody (ΔPrp24) or 5 μl of phosphate-buffered saline (mock-depleted) on ice for 1 hour and then nutated for 1 hour with ∼20 μl of protein A-Sepharose beads (Pharmacia) washed with buffer D [20 mM Hepes (pH 7.9), 0.2 mM EDTA, 50 mM KCl, 20% (v/v) glycerol, 0.5 mM dithiothreitol (DTT)]. The depleted supernatants lacked Prp24 and free U6 snRNP (Fig. 1A, lanes 7 to 10).
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24. 2). Electrophoresis was for 6 to 7 hours at 160 V in TGM buffer at 4°C. Northern analysis was conducted as described (33). To supershift snRNP complexes formed during 23°C incubation, samples were incubated with 1 μg of 12CA5 antibody for 1 hour on ice before loading.
25. P. L. Raghunathan and C. Guthrie, unpublished results.
26. The first 220 bp of the PRP24 gene were PCR amplified as a Bam HI-Apa I fragment (using an internal Apa I site and a primer with a Bam HI site just upstream of the start codon). This fragment was inserted downstream of the GPD (glyceraldehyde-3-phosphate dehydrogenase) promoter of pG1 [M. Schena, D. Picard, K. R. Yamamoto, Methods Enzymol. 194, 389 (1991)] along with either a 1.6-kb Apa I-Sal I PRP24 fragment or a 1.4-kb Apa I-Sal I PRP24-Pya fragment (13). Both constructs overexpressed Prp24p when transformed into PRY98 as determined by Western blotting with anti-Prp24 polyclonal antibodies.
27. 2, 10% glycerol, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, pepstatin A (1 μg/ml), leupeptin (1 μg/ml)] and incubated with 150 μl of protein G-Sepharose (washed in AGK 200) on a nutator at 4°C for 30 min. (This step removed proteins that bound nonspecifically to the resin.) The supernatants were added to 1 ml of protein G-Sepharose coupled to anti-polyoma antibodies (beads: AGK 200 = 1:1) as in (32). After 1.5 hours nutating at 4°C, the supernatants were removed, and the beads were washed 5 × 20 ml of AGK 700 (AGK buffer with 700 mM KCl), 1 × 15 ml of AGK 200, and 1 × 15 ml of AGK 50 (AGK buffer with 50 mM KCl). The washed beads were eluted twice by nutating at 23°C for 10 min with 2 × 400 μl (1 mg/ml) of peptide EYMPME (Glu-Tyr-Met-Pro-Met-Glu) (32) in AGK 50. Each eluate was dialyzed against 2 × 2 liters of buffer D at 4°C for 3.5 hours. The dialyzates were microcentrifuged 10 min, and the supernatants were collected. Approximately 15 μg of Prp24 was recovered, and no snRNAs copurified under these conditions (with a sensitivity to ∼1 fmol of U6, no U6 was detected in 1 pmol of Prp24 protein).
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38. Splicing reaction mixtures (80 μl) (22) containing extract, ATP, and unlabeled actin pre-mRNA as noted were incubated 30 min at 25°C. After nutating for 1 hour at 4°C with 3.2 μg of 12CA5 antibody and 400 μl of NET 50 [50 mM tris-HCl (pH 7.4), 50 mM NaCl, 0.05% NP-40], antibody complexes were mixed with protein A-Sepharose (Pharmacia) for 1 hour at 4°C. The bound complexes were washed three times with 800 μl of NET 50, extracted on ice twice with phenol chloroform, and ethanol-precipitated to isolate RNAs.
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50. 2, 3% PEG 8000, 60 mM potassium phosphate, with or without 2 mM ATP). The supernatants were separated from the beads, and the beads were washed with 1 × 500 μl of NET 50. For Fig. 4A, the samples were directly deproteinized for nondenaturing gel analysis of U4 and U6 snRNAs. For Fig. 4, B and C, 1 μl of purified Prp24 was added to 11 μl of supernatant containing ATP (∼0.4 nM each U4 and U6) and incubated for 2 min at 23°C unless otherwise stated. Reactions were stopped by the addition of 200 μl of 0.3 M sodium acetate, 6 mM EDTA, and 0.5% SDS on ice. RNAs were isolated by proteinase K digestion (3) or by phenol chloroform extraction and ethanol precipitation. To deproteinize U4 and U6 snRNPs before annealing, 100 μl of supernatant containing ATP was incubated at 23°C for 30 min with 10 mg of proteinase K beads (Sigma) washed in buffer D. The deproteinized supernatant was carefully collected for annealing reactions.
51. We thank A. Ghetti and J. Abelson for recombinant Prp24 protein; M. Lenburg, B. O'Neill, and E. Q'Shea for advice on immunoaffinity purification; J. Brown for the anti-polyoma hybridoma line; C. Collins and S. Rader for advice and assistance; past and present members of the Guthrie laboratory for constructive criticism; and E. Blackburn, C. Collins, A. Frankel, H. Madhani, S. Rader, C. Siebel, J. Staley, O. Uhlenbeck, and K. Zingler for comments on the manuscript. We are indebted to L. Esperas, C. Pudlow, and H. Roiha for matchless technical assistance. P.L.R. was supported by a Howard Hughes Medical Institute predoctoral fellowship. G.G. is an American Cancer Society Research Professor of Molecular Genetics. Supported by NIH grant GM21119 to C.G.