Identification of both shared and distinct proteins in the major and minor spliceosomes

Science

Volumn 284, Issue 5422, 1999, Pages 2003-2005

  Will, Cindy L ^a   Schneider, Claudia ^a   Reed, Robin ^b   Lührmann, Reinhard ^a,c  

^a PHILIPPS UNIVERSITY MARBURG   (Germany)

^b HARVARD MEDICAL SCHOOL   (United States)

^c MAX PLANCK INSTITUTE FOR BIOPHYSICAL CHEMISTRY   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

POLYPEPTIDE; SMALL NUCLEAR RIBONUCLEOPROTEIN;

ARTICLE; EVOLUTION; IMMUNOBLOTTING; INTRON; METAZOON; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN ISOLATION; RNA SPLICING; SPLICEOSOME;

AMINO ACID SEQUENCE; CHROMATOGRAPHY, AFFINITY; EVOLUTION, MOLECULAR; HELA CELLS; HUMANS; INTRONS; MOLECULAR SEQUENCE DATA; MOLECULAR WEIGHT; RIBONUCLEOPROTEIN, U1 SMALL NUCLEAR; RIBONUCLEOPROTEIN, U2 SMALL NUCLEAR; RIBONUCLEOPROTEINS, SMALL NUCLEAR; RNA SPLICING; SPLICEOSOMES;

METAZOA;

EID: 0037634375     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5422.2003     Document Type: Article

References (37)

1. C. B. Burge, T. H. Tuschl, P. A. Sharp, in The RNA World II, R. F. Gesteland and J. F. Atkins, Eds. (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1999), pp. 525-560.
2. S. L. Hall and R. A. Padgett, J. Mol. Biol. 239, 357 (1994); P. A. Sharp and C. B. Burge, Cell 91, 875 (1997).
3. S. L. Hall and R. A. Padgett, J. Mol. Biol. 239, 357 (1994); P. A. Sharp and C. B. Burge, Cell 91, 875 (1997).
4. S. L. Hall and R. A. Padgett, Science 271, 1716 (1996); W.-Y. Tarn and J. A. Steitz, Cell 84, 801 (1996); Y.-T. Yu and J. A. Steitz, Proc. Natl. Acad. Sci. U.S.A. 94, 6030 (1997); I. Kolossova and R. A. Padgett, RNA 3, 227 (1997).
5. S. L. Hall and R. A. Padgett, Science 271, 1716 (1996); W.-Y. Tarn and J. A. Steitz, Cell 84, 801 (1996); Y.-T. Yu and J. A. Steitz, Proc. Natl. Acad. Sci. U.S.A. 94, 6030 (1997); I. Kolossova and R. A. Padgett, RNA 3, 227 (1997).
6. S. L. Hall and R. A. Padgett, Science 271, 1716 (1996); W.-Y. Tarn and J. A. Steitz, Cell 84, 801 (1996); Y.-T. Yu and J. A. Steitz, Proc. Natl. Acad. Sci. U.S.A. 94, 6030 (1997); I. Kolossova and R. A. Padgett, RNA 3, 227 (1997).
7. S. L. Hall and R. A. Padgett, Science 271, 1716 (1996); W.-Y. Tarn and J. A. Steitz, Cell 84, 801 (1996); Y.-T. Yu and J. A. Steitz, Proc. Natl. Acad. Sci. U.S.A. 94, 6030 (1997); I. Kolossova and R. A. Padgett, RNA 3, 227 (1997).
8. W.-Y. Tarn and J. A. Steitz, Science 273, 1824 (1996).
9. R. Reed, Curr Opin. Genet. Dev. 6, 215 (1996).
10. C. L. Will and R. Lührmann, Curr. Opin. Cell Biol. 9, 320 (1997).
11. A. Krämer, Annu. Rev. Blochem. 65, 367 (1996).
12. K. M. Wassarman and J. A. Steitz, Mol. Cell. Biol. 8, 1276 (1992).
13. M. Frilander and J. A. Steitz, Genes Dev. 13, 851 (1999).
14. Trimethylguanosine (m3G)-capped snRNPs were immunoaffinity-purified from HeLa nuclear extracts with antibodies against m3G and separated on 10 to 30% glycerol gradients [B. Uggerbauer, J. Lauber, R. Lührmann, Nucleic Acids Res. 24, 368 (1996)]. Fractions containing 185 U11/U12 snRNP complexes were pooled and the KCl concentration was adjusted to 250 mM. snRNPs from 2.4 ml of pooled 18S gradient fractions were incubated for 16 hours at 4°C with 12 μg of an oligonucleotide complementary to nucleotides 2 to 18 of human U11 snRNA, 5′-ACGACAGAAGCCCUUUUdT*dT*dT'dr-3′ (U11 oligo), or complementary to nucleotides 11 to 28 of human U12 snRNA, 5′-AUUUUCCUUACUCAUAAGdrdT*dT*dT*-3′ (U12 oligo), where * denotes a biotinylated 2′-deoxythymidine and A, U, G, and C represent 2′-O-methyl ribonucleotides. Oligonucleotide-bound snRNPs were precipitated with streptavidin-agarose [A. I. Lamond and B. S. Sproat, in RNA Processing: A Practical Approach, D. Rickwood and B. D. Harnes, Eds. (Oxford Univ. Press, Oxford, 1996), pp. 103-140]. RNA was eluted from one-fifth of the agarose-precipitated snRNPs by incubating for 30 min at 95°C in 100 μl of DH buffer (15 mM NaCl, 1.5 mM Na citrate, and 0.1% SDS), fractionated on 10% polyacrylamide-7 M urea gels, and visualized by silver staining, The Identity of the selected RNAs as U11 and U12 was confirmed by Northern blotting. Protein was eluted from the remaining beads by incubating for S min at 95°C in 200 μ of S buffer [60 mM tris (pH 6.8), 1 mM EDTA. 17.5% glycerol. 2% SDS, and 0.2 M dithioerythritol (DTE)] and precipitated with five volumes of acetone. Proteins were fractionated by SDS-polyacryiamide gel electrophoresis (PACE) on gels containing 10% (upper halt) and 13% (lower half) polyacrylamide, and visualized by Coomassie staining. For comparison, RNA and protein from 50 μl of the input material (pooled 185 gradient fractions) were also analyzed
15. Trimethylguanosine (m3G)-capped snRNPs were immunoaffinity-purified from HeLa nuclear extracts with antibodies against m3G and separated on 10 to 30% glycerol gradients [B. Uggerbauer, J. Lauber, R. Lührmann, Nucleic Acids Res. 24, 368 (1996)]. Fractions containing 185 U11/U12 snRNP complexes were pooled and the KCl concentration was adjusted to 250 mM. snRNPs from 2.4 ml of pooled 18S gradient fractions were incubated for 16 hours at 4°C with 12 μg of an oligonucleotide complementary to nucleotides 2 to 18 of human U11 snRNA, 5′-ACGACAGAAGCCCUUUUdT*dT*dT'dr-3′ (U11 oligo), or complementary to nucleotides 11 to 28 of human U12 snRNA, 5′-AUUUUCCUUACUCAUAAGdrdT*dT*dT*-3′ (U12 oligo), where * denotes a biotinylated 2′-deoxythymidine and A, U, G, and C represent 2′-O-methyl ribonucleotides. Oligonucleotide-bound snRNPs were precipitated with streptavidin-agarose [A. I. Lamond and B. S. Sproat, in RNA Processing: A Practical Approach, D. Rickwood and B. D. Harnes, Eds. (Oxford Univ. Press, Oxford, 1996), pp. 103-140]. RNA was eluted from one-fifth of the agarose-precipitated snRNPs by incubating for 30 min at 95°C in 100 μl of DH buffer (15 mM NaCl, 1.5 mM Na citrate, and 0.1% SDS), fractionated on 10% polyacrylamide-7 M urea gels, and visualized by silver staining, The Identity of the selected RNAs as U11 and U12 was confirmed by Northern blotting. Protein was eluted from the remaining beads by incubating for S min at 95°C in 200 μ of S buffer [60 mM tris (pH 6.8), 1 mM EDTA. 17.5% glycerol. 2% SDS, and 0.2 M dithioerythritol (DTE)] and precipitated with five volumes of acetone. Proteins were fractionated by SDS-polyacryiamide gel electrophoresis (PACE) on gels containing 10% (upper halt) and 13% (lower half) polyacrylamide, and visualized by Coomassie staining. For comparison, RNA and protein from 50 μl of the input material (pooled 185 gradient fractions) were also analyzed
16. C. L. Will and R. Lührmann, in Eukaryotic mRNA Processing, A. R. Krainer, Ed. (IRL Press, Oxford, 1997). p. 130.
17. C. L. Will, C. Schneider, R. Reed, R. Lührmann, unpublished data.
18. The difference in the apparent molecular mass of some of the comigrating proteins (such as U2-53RD and U11/U12-49kD) is due to differences in the electrophoresis conditions originally used to identify the 17S U2 snRNP proteins [S.-E. Behrens et al., Mol. Cell. Biol. 13, 307 (1993)]. Note that U2-160kD, U2-150kD, U2-120kD, and U2-53kD also correspond to the spliceosomal associated proteins (SAPs) of 155, 145, 130. and 49 kD, respectively.
19. Sequencing of tryptic peptides generated from each protein was performed by Toplab (Martinsried, Germany).
20. A database search with the U11/U12-130kD peptide containing three mismatches did not detect, among a total of 36 positives, any expressed sequence tags (ESTs) whose open reading frame (ORF) contained one or more of the observed amino acid changes. This indicates that U11/U12-130kD is probably not a splice variant of the U2-120kD protein; rather, these discrepancies most likely represent peptide sequencing errors.
21. C. Wang et al., Genes Dev. 12, 1409 (1998).
22. O. Gozani, R. Feld, R. Reed, ibid. 10, 233 (1996).
23. P. Champion-Arnaud and R. Reed, ibid. 8, 1974 (1994).
24. N. Abovich and M. Rosbash, Cell 89, 403 (1997); J. A. Berglund, K. Chua, N. Abovich, R. Reed, M. Rosbash, ibid., p. 781; M. T. Bedford, R. Reed. P. Leder, Proc. Nati. Acad. Sci. U.S.A. 95, 10602 (1998).
25. N. Abovich and M. Rosbash, Cell 89, 403 (1997); J. A. Berglund, K. Chua, N. Abovich, R. Reed, M. Rosbash, ibid., p. 781; M. T. Bedford, R. Reed. P. Leder, Proc. Nati. Acad. Sci. U.S.A. 95, 10602 (1998).
26. N. Abovich and M. Rosbash, Cell 89, 403 (1997); J. A. Berglund, K. Chua, N. Abovich, R. Reed, M. Rosbash, ibid., p. 781; M. T. Bedford, R. Reed. P. Leder, Proc. Nati. Acad. Sci. U.S.A. 95, 10602 (1998).
27. Peptide sequences have been obtained for all but the 28.5-and 20-kD U11/U12 proteins. Antibodies specific for the U1-70K, U1-A, U1-C, or U2-B″ proteins did not recognize any of the U11/U12-associated proteins. Because the sequences of the U2-35kD and U2-92kD proteins are currently unknown, we cannot rigorously exclude that similar proteins are present in the U11/U12 complex. However, antibodies directed against the U11/U12-3SkD protein did not recognize U2-35kD on immunoblots.
28. Peptide sequences were obtained from gel-fractionated, trypsin-digested U11/U12 proteins by microsequencing. The 35-kD protein-peptides KEYDPLK and KRWQEREPTRVWPDND (28) were used to search the National Center for Biotechnology Information EST database for full-length cDNAs by means of the program TBLASTN. Both peptides were found In the ORF of a cDNA from human macrophage cells (Gen-Bank accession number U44798) encoding a protein of unknown function that was noted to share homology with the U 1-70K protein. An EST cONA from human muscle cells that contains an identical ORF (GenBank accession number AA211268) was cloned and sequenced in its entirety. Protein generated by in vitro translation of this cDNA comigrated with the purified U11/U12-35kD protein on SDS-polyacrylamide gels, confirming that it encodes full-length protein (72).
29. C. B. Burge, R. A. Padgett, P. A. Sharp, Mol. Cell 2, 773 (1998).
30. See Science Online (www.sclencemag.org/feature/ data/990628.shl) for sequence alignment of the U11/ U12-35kD and U1-70K proteins.
31. K. M. Neugebauer, J. A. Stolk, M. B. Roth, J. Cell Biol. 129, 899 (1995).
32. The U11/U12-35kD protein peptide EKRWQEREPTRVWPD (positions 208 to 222) (28) was used to generate rabbit antibodies that reacted specifically with the 35-kD protein on immunoblots (72). Immunoprecipitations were performed as described [S. Teigelkamp, C. Mundt, T. Achsel, C. L. Will, R. Lührmann, RNA 3, 1313 (1997)], except gradient-fractionated 12S snRNPs were used as the input material.
33. Q. Wu and A. R. Krainer, Science 274, 1005 (1996).
34. C. Schneider and R. Lührmann, unpublished data.
35. Abbreviations for amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
36. B. Das, L. Xia, O. Gozani, L. Palandjian, Y. Chyung, R. Reed, unpublished date.
37. We thank G. Heyne, A. Badouin, W. Lorenz, D. Meyer, and I. Öchsner for excellent technical assistance, M. Krause for the synthesis of 2′-O-methyl oligonucleotides, and the Resource Center of the German Genome Project at the Max Planck Institute for Molecular Genetics, as welt as the IMAGE cDNA Clone Consortium, for providing EST clones. Supported by the Gottfried Wilhelm Leibniz Program and grants from the Deutsche Forschungsgemeinschaft (A6/ SFB397) and Fonds der Chemischen Industrie (R.L).