U1-mediated exon definition interactions between AT-AC and GT-AG introns

Science

Volumn 274, Issue 5289, 1996, Pages 1005-1008

  Wu, Qiang ^a   Krainer, Adrian R ^a  

^a Howard Hughes Medical Institute Cold Spring Harbor Laboratory Cold Spring Harbor   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ADENOSINETRIPHOSPHATE; CATALYSIS; CELLS; DISEASES; ENZYMES; GENES; MAGNESIUM; PROTEINS;

EXONS; GENE EXPRESSION; METAZOAN INTRONS; NUCLEAR RIBONUCLEOPROTEIN; NUCLEOTIDES; SPLICEOSOMES;

RNA;

ADENOSINE TRIPHOSPHATE; MAGNESIUM; MESSENGER RNA; RIBONUCLEASE H; SMALL NUCLEAR RNA; SODIUM CHANNEL;

ARTICLE; EXON; FLY; GENE EXPRESSION REGULATION; HUMAN; INTRON; MOUSE; NONHUMAN; POINT MUTATION; PRIORITY JOURNAL; RNA SPLICING; SPLICEOSOME;

ADENOSINE TRIPHOSPHATE; ANIMALS; EXONS; HELA CELLS; HUMANS; INTRONS; MAGNESIUM; RIBONUCLEOPROTEIN, U1 SMALL NUCLEAR; RIBONUCLEOPROTEINS, SMALL NUCLEAR; RNA SPLICING; RNA, SMALL NUCLEAR; SODIUM CHANNELS; SPLICEOSOMES;

METAZOA;

EID: 0030575916     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5289.1005     Document Type: Article

References (61)

1. M. J. Moore, C. C. Query, P. A. Sharp, in The RNA World, R. F. Gesteland and J. F. Atkins, Eds. (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1993), pp. 303-358; T. W. Nilsen, Cell 78, 1 (1994); H. D. Madhani and C. Guthrie, Annu Rev. Genet. 28, 1 (1994).
2. M. J. Moore, C. C. Query, P. A. Sharp, in The RNA World, R. F. Gesteland and J. F. Atkins, Eds. (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1993), pp. 303-358; T. W. Nilsen, Cell 78, 1 (1994); H. D. Madhani and C. Guthrie, Annu Rev. Genet. 28, 1 (1994).
3. M. J. Moore, C. C. Query, P. A. Sharp, in The RNA World, R. F. Gesteland and J. F. Atkins, Eds. (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1993), pp. 303-358; T. W. Nilsen, Cell 78, 1 (1994); H. D. Madhani and C. Guthrie, Annu Rev. Genet. 28, 1 (1994).
4. M. Krawczak, J. Reiss, D. N. Cooper, Hum. Genet. 90, 41 (1992); K. Naka and H. Sakamoto, Gene 141, 171 (1994).
5. M. Krawczak, J. Reiss, D. N. Cooper, Hum. Genet. 90, 41 (1992); K. Naka and H. Sakamoto, Gene 141, 171 (1994).
6. R. Parker and P. G. Siliciano, Nature 361, 660 (1993); G. Chanfreau, P. Legrain, B. Dujon, A. Jacquier, Nucleic Acids Res. 22, 1981 (1994) ; A. Deirdre, J. Scadden, C. W. Smith. EMBO J. 14, 3236 (1995).
7. R. Parker and P. G. Siliciano, Nature 361, 660 (1993); G. Chanfreau, P. Legrain, B. Dujon, A. Jacquier, Nucleic Acids Res. 22, 1981 (1994) ; A. Deirdre, J. Scadden, C. W. Smith. EMBO J. 14, 3236 (1995).
8. R. Parker and P. G. Siliciano, Nature 361, 660 (1993); G. Chanfreau, P. Legrain, B. Dujon, A. Jacquier, Nucleic Acids Res. 22, 1981 (1994) ; A. Deirdre, J. Scadden, C. W. Smith. EMBO J. 14, 3236 (1995).
9. I. J. Jackson, Nucleic Acids Res. 19, 3795 (1991).
10. S. L. Hall and R. A. Padgett, J. Mol. Biol. 139, 357 (1994).
11. I. Kiss et al., J. Biol. Chem. 264, 8126 (1989); R. N. Jenkins et al., ibid. 265, 19624 (1990); R. G. Larson et al., Cancer Commun. 2, 63 (1990); A. I. McClatchey et al., Hum. Mol. Genet. 1, 521 (1992); A. L. George Jr., G. S. Iyer, R. Kleinfield, R.G. Kallen, R. L. Barchi, Genomics 15, 598 (1993); T. Ohshima et al., ibid. 28, 585 (1995); Q. Wang, Z. Li, J. Shen, M. T. Keating, ibid. 34, 9 (1996).
12. I. Kiss et al., J. Biol. Chem. 264, 8126 (1989); R. N. Jenkins et al., ibid. 265, 19624 (1990); R. G. Larson et al., Cancer Commun. 2, 63 (1990); A. I. McClatchey et al., Hum. Mol. Genet. 1, 521 (1992); A. L. George Jr., G. S. Iyer, R. Kleinfield, R.G. Kallen, R. L. Barchi, Genomics 15, 598 (1993); T. Ohshima et al., ibid. 28, 585 (1995); Q. Wang, Z. Li, J. Shen, M. T. Keating, ibid. 34, 9 (1996).
13. I. Kiss et al., J. Biol. Chem. 264, 8126 (1989); R. N. Jenkins et al., ibid. 265, 19624 (1990); R. G. Larson et al., Cancer Commun. 2, 63 (1990); A. I. McClatchey et al., Hum. Mol. Genet. 1, 521 (1992); A. L. George Jr., G. S. Iyer, R. Kleinfield, R.G. Kallen, R. L. Barchi, Genomics 15, 598 (1993); T. Ohshima et al., ibid. 28, 585 (1995); Q. Wang, Z. Li, J. Shen, M. T. Keating, ibid. 34, 9 (1996).
14. I. Kiss et al., J. Biol. Chem. 264, 8126 (1989); R. N. Jenkins et al., ibid. 265, 19624 (1990); R. G. Larson et al., Cancer Commun. 2, 63 (1990); A. I. McClatchey et al., Hum. Mol. Genet. 1, 521 (1992); A. L. George Jr., G. S. Iyer, R. Kleinfield, R.G. Kallen, R. L. Barchi, Genomics 15, 598 (1993); T. Ohshima et al., ibid. 28, 585 (1995); Q. Wang, Z. Li, J. Shen, M. T. Keating, ibid. 34, 9 (1996).
15. I. Kiss et al., J. Biol. Chem. 264, 8126 (1989); R. N. Jenkins et al., ibid. 265, 19624 (1990); R. G. Larson et al., Cancer Commun. 2, 63 (1990); A. I. McClatchey et al., Hum. Mol. Genet. 1, 521 (1992); A. L. George Jr., G. S. Iyer, R. Kleinfield, R.G. Kallen, R. L. Barchi, Genomics 15, 598 (1993); T. Ohshima et al., ibid. 28, 585 (1995); Q. Wang, Z. Li, J. Shen, M. T. Keating, ibid. 34, 9 (1996).
16. I. Kiss et al., J. Biol. Chem. 264, 8126 (1989); R. N. Jenkins et al., ibid. 265, 19624 (1990); R. G. Larson et al., Cancer Commun. 2, 63 (1990); A. I. McClatchey et al., Hum. Mol. Genet. 1, 521 (1992); A. L. George Jr., G. S. Iyer, R. Kleinfield, R.G. Kallen, R. L. Barchi, Genomics 15, 598 (1993); T. Ohshima et al., ibid. 28, 585 (1995); Q. Wang, Z. Li, J. Shen, M. T. Keating, ibid. 34, 9 (1996).
17. I. Kiss et al., J. Biol. Chem. 264, 8126 (1989); R. N. Jenkins et al., ibid. 265, 19624 (1990); R. G. Larson et al., Cancer Commun. 2, 63 (1990); A. I. McClatchey et al., Hum. Mol. Genet. 1, 521 (1992); A. L. George Jr., G. S. Iyer, R. Kleinfield, R.G. Kallen, R. L. Barchi, Genomics 15, 598 (1993); T. Ohshima et al., ibid. 28, 585 (1995); Q. Wang, Z. Li, J. Shen, M. T. Keating, ibid. 34, 9 (1996).
18. The published 5′ splice site sequence of intron 2 of human CACNL1A1, the fibroblast voltage-gated L-type calcium channel, precisely matches the AT-AC consensus, whereas the reported sequences of two other calcium channels, CACNL1A2 and CACNL1A3, have GTATCC (rather than ATATCC) at the corresponding 5′ splice site; however, all three calcium channel introns reportedly end with the conventional AG 3′ splice site [N. M. Soldatov, Genomics 22, 77 (1994); Y. Yamada et al., ibid. 27, 312 (1995); K. Hogan, R. G. Gregg, P. A. Powers, ibid. 31, 392 (1996)].
19. The published 5′ splice site sequence of intron 2 of human CACNL1A1, the fibroblast voltage-gated L-type calcium channel, precisely matches the AT-AC consensus, whereas the reported sequences of two other calcium channels, CACNL1A2 and CACNL1A3, have GTATCC (rather than ATATCC) at the corresponding 5′ splice site; however, all three calcium channel introns reportedly end with the conventional AG 3′ splice site [N. M. Soldatov, Genomics 22, 77 (1994); Y. Yamada et al., ibid. 27, 312 (1995); K. Hogan, R. G. Gregg, P. A. Powers, ibid. 31, 392 (1996)].
20. The published 5′ splice site sequence of intron 2 of human CACNL1A1, the fibroblast voltage-gated L-type calcium channel, precisely matches the AT-AC consensus, whereas the reported sequences of two other calcium channels, CACNL1A2 and CACNL1A3, have GTATCC (rather than ATATCC) at the corresponding 5′ splice site; however, all three calcium channel introns reportedly end with the conventional AG 3′ splice site [N. M. Soldatov, Genomics 22, 77 (1994); Y. Yamada et al., ibid. 27, 312 (1995); K. Hogan, R. G. Gregg, P. A. Powers, ibid. 31, 392 (1996)].
21. The sodium and calcium channels are thought to derive from a common ancestral gene: they have considerable nucleotide and amino acid sequence homology. and the unusual intron interrupts a homologous position of the coding sequence in all five genes. Unless there are errors in some of the reported sequences, it will be interesting to determine whether the calcium channel transcripts are processed via the major pathway, the AT-AC pathway, or a hybird pathway.
22. 8. A portion of the human SCN4A gene was amplified by PCR from human total genomic DNA (Promega) with primers containing restriction sites and matching exons 2 and 3, to generate a fragment comprising nt 886 to 1229 (numbering according to Gen-Bank accession number L04216). This fragment was digested with Hind III and Xba I and subcloned into the corresponding sites of pSP64 (Promega) to generate the pSP64-SCN4A plasmid. For construction of pSP64-SCN4AM, a different downstream PCR primer containing the mutations and an Eco RI site was used to amplify a mutant fragment from the cloned wild-type template, which was then subcloned as a Hind III-Eco RI fragment in pSP64. All constructs were confirmed by sequence analysis. pSP64-SCN4A and pSP64-SCN4AM were linearized with Xba I or Eco RI, respectively, for use as templates for in vitro transcription with SP6 RNA polymerase. The transcripts contain short extensions at both ends, derived from the vector.
23. 2 was omitted. RNA was recovered and analyzed on 4.5% denaturing polyacrylamide gels, followed by autoradiography. Splicing efficiency, defined as the molar ratio mRNA/(pre-mRNA + mRNA). was estimated by phosphor image analysis (Fujix, BAS2000).
24. Q. Wu and A. R. Krainer, data not shown.
25. To sequence the authentic and cryptic spliced mRNAs across the spliced junctions, we amplified each gel-purified RNA by RT-PCR with exon 2 and exon 3 primers (TCATCGTACTCAACAAGG and TACTCCACATTCTTGGAC). The amplified fragment, subcloned into PCR2.1 (Invitrogen), was sequenced with T7 Sequenase 2.0 (USB).
26. P. J. Grabowski, R. A. Padgett, P. A. Sharp, Cell 37, 415 (1984); B. Ruskin, A. R. Krainer, T. Maniatis, M. R. Green, ibid. 38, 317 (1984).
27. P. J. Grabowski, R. A. Padgett, P. A. Sharp, Cell 37, 415 (1984); B. Ruskin, A. R. Krainer, T. Maniatis, M. R. Green, ibid. 38, 317 (1984).
28. W.-Y. Tarn and J. A. Steitz, ibid. 84, 801 (1996).
29. A. Krämer, W. Keller, B. Appel, R. Lührmann, ibid. 38, 299 (1984); A. R. Krainer and T. Maniatis. ibid. 42, 725 (1985); D. L. Black, B. Chabot, J. A. Steitz, ibid., p. 737; S. M. Berget and B. L. Robberson, ibid. 46, 691 (1986); D. L. Black and J. A. Steitz, ibid., p. 697.
30. A. Krämer, W. Keller, B. Appel, R. Lührmann, ibid. 38, 299 (1984); A. R. Krainer and T. Maniatis. ibid. 42, 725 (1985); D. L. Black, B. Chabot, J. A. Steitz, ibid., p. 737; S. M. Berget and B. L. Robberson, ibid. 46, 691 (1986); D. L. Black and J. A. Steitz, ibid., p. 697.
31. A. Krämer, W. Keller, B. Appel, R. Lührmann, ibid. 38, 299 (1984); A. R. Krainer and T. Maniatis. ibid. 42, 725 (1985); D. L. Black, B. Chabot, J. A. Steitz, ibid., p. 737; S. M. Berget and B. L. Robberson, ibid. 46, 691 (1986); D. L. Black and J. A. Steitz, ibid., p. 697.
32. A. Krämer, W. Keller, B. Appel, R. Lührmann, ibid. 38, 299 (1984); A. R. Krainer and T. Maniatis. ibid. 42, 725 (1985); D. L. Black, B. Chabot, J. A. Steitz, ibid., p. 737; S. M. Berget and B. L. Robberson, ibid. 46, 691 (1986); D. L. Black and J. A. Steitz, ibid., p. 697.
33. A. Krämer, W. Keller, B. Appel, R. Lührmann, ibid. 38, 299 (1984); A. R. Krainer and T. Maniatis. ibid. 42, 725 (1985); D. L. Black, B. Chabot, J. A. Steitz, ibid., p. 737; S. M. Berget and B. L. Robberson, ibid. 46, 691 (1986); D. L. Black and J. A. Steitz, ibid., p. 697.
34. For RNase H inhibition experiments, the nuclear extract was preincubated for 15 min under splicing conditions in the presence or absence of the appropriate oligonucleotides. The oligonucleotides were complementary to U1 snRNA position 2 to 11, U2 snRNA position 1 to 15, or U12 snRNA position 11 to 24, snRNA cleavage is catalyzed by the endogenous RNase H (14), and exogenous RNase H had no additional effect (10). All snRNA cleavage and splicing inhibition experiments were carried out at least three times, with reproducible results.
35. S. J. Baserga and J. A. Steitz, in The RNA World, R. F. Gesteland and J. F. Atkins, Eds. (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1993), pp. 359-381; D. A. Wassarman and J. A. Steitz, Mol. Cell. Biol. 11, 3432 (1991).
36. S. J. Baserga and J. A. Steitz, in The RNA World, R. F. Gesteland and J. F. Atkins, Eds. (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1993), pp. 359-381; D. A. Wassarman and J. A. Steitz, Mol. Cell. Biol. 11, 3432 (1991).
37. S. L. Hall and R. A. Padgett, Science 271, 1716 (1996).
38. K. A. Montzka and J. A. Steitz, Proc. Natl. Acad. Sci. U.S.A. 85, 8885 (1988); D. L. Black and A. L. Pinto, Mol. Cell. Biol. 9, 3350 (1989).
39. K. A. Montzka and J. A. Steitz, Proc. Natl. Acad. Sci. U.S.A. 85, 8885 (1988); D. L. Black and A. L. Pinto, Mol. Cell. Biol. 9, 3350 (1989).
40. K. M. Wasserman and J. A. Steitz, Mol. Cell. Biol. 12, 1276 (1992).
41. R. R. Gontarek, M. T. McNally, K. Beemon, Genes Dev. 7, 1926 (1993).
42. G. P. Shumyatsky, S. V. Tillib, D. A. Kramerov, Nucleic Acids Res. 18, 6347 (1990).
43. Y. Zhuang and A. M. Weiner, Cell 46, 827 (1986); P. G. Siliciano and C. Guthrie, Genes Dev. 2, 1258 (1988); B. Seraphin, L. Kretzner, M. Rosbash, EMBO J. 7, 1533 (1988).
44. Y. Zhuang and A. M. Weiner, Cell 46, 827 (1986); P. G. Siliciano and C. Guthrie, Genes Dev. 2, 1258 (1988); B. Seraphin, L. Kretzner, M. Rosbash, EMBO J. 7, 1533 (1988).
45. Y. Zhuang and A. M. Weiner, Cell 46, 827 (1986); P. G. Siliciano and C. Guthrie, Genes Dev. 2, 1258 (1988); B. Seraphin, L. Kretzner, M. Rosbash, EMBO J. 7, 1533 (1988).
46. B. L. Robberson, G. J. Cote, S. M. Berget, Mol. Cell. Biol. 10, 84 (1990); H.-C. Kuo, F. H. Nasim, P. J. Grabowski, Science 251, 1045 (1991); J. P. Kreivi, K. Zerivitz, G. Akusjärvi, Nucleic Acids Res. 19, 6956 (1991).
47. B. L. Robberson, G. J. Cote, S. M. Berget, Mol. Cell. Biol. 10, 84 (1990); H.-C. Kuo, F. H. Nasim, P. J. Grabowski, Science 251, 1045 (1991); J. P. Kreivi, K. Zerivitz, G. Akusjärvi, Nucleic Acids Res. 19, 6956 (1991).
48. B. L. Robberson, G. J. Cote, S. M. Berget, Mol. Cell. Biol. 10, 84 (1990); H.-C. Kuo, F. H. Nasim, P. J. Grabowski, Science 251, 1045 (1991); J. P. Kreivi, K. Zerivitz, G. Akusjärvi, Nucleic Acids Res. 19, 6956 (1991).
49. The SCN4AM mRNA was accurately spliced, as verified by RT-PCR sequencing. No spliced products were detected in the absence of ATP or magnesium. The SCN4AM cryptic spliced product was also confirmed by sequencing. Cleavage of U4 and U6 snRNAs had the same effect as U2 cleavage. An additional pre-mRNA, in which the downstream 5′ splice site was deleted, had identical splicing and inhibition profiles as those of the SCN4AM substrate (10).
50. S. M. Berget, J. Biol. Chem. 270, 2411 (1995).
51. M. D. Chiara and R. Reed, Nature 375, 510 (1995).
52. M. Ohno, H. Sakamoto, Y. Shimura, Proc. Natl. Acad. Sci. U.S.A. 84, 5187 (1987); E. Izaurralde et al., Cell 78, 657 (1994).
53. M. Ohno, H. Sakamoto, Y. Shimura, Proc. Natl. Acad. Sci. U.S.A. 84, 5187 (1987); E. Izaurralde et al., Cell 78, 657 (1994).
54. M. Niwa and S. M. Berget, Genes Dev. 5, 2086 (1991); K. M. Wassarman and J. A. Steitz, ibid. 7, 647 (1993); C. S. Lutz et al., ibid. 10, 325 (1996).
55. M. Niwa and S. M. Berget, Genes Dev. 5, 2086 (1991); K. M. Wassarman and J. A. Steitz, ibid. 7, 647 (1993); C. S. Lutz et al., ibid. 10, 325 (1996).
56. M. Niwa and S. M. Berget, Genes Dev. 5, 2086 (1991); K. M. Wassarman and J. A. Steitz, ibid. 7, 647 (1993); C. S. Lutz et al., ibid. 10, 325 (1996).
57. B. E. Hoffman and P. J. Grabowski, ibid. 6, 2554 (1992); J. Y. Wu and T. Maniatis, Cell 75, 1061 (1993); D. Staknis and R. Reed, Mol. Cell. Biol. 14, 7670 (1994).
58. B. E. Hoffman and P. J. Grabowski, ibid. 6, 2554 (1992); J. Y. Wu and T. Maniatis, Cell 75, 1061 (1993); D. Staknis and R. Reed, Mol. Cell. Biol. 14, 7670 (1994).
59. B. E. Hoffman and P. J. Grabowski, ibid. 6, 2554 (1992); J. Y. Wu and T. Maniatis, Cell 75, 1061 (1993); D. Staknis and R. Reed, Mol. Cell. Biol. 14, 7670 (1994).
60. D. C. Kohrmann, J. B. Harris, M. H. Meisler, J. Biol. Chem. 271, 17576 (1996).
61. We thank D. Horowitz for comments on the manuscript; A. Mayeda for sharing protocols and reagents; and M. Zhang, M. Murray, H.-X. Liu, and T.-L. Tseng for helpful discussions. Supported by grant GM42699 from NIH.