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. 1997 Mar 1;25(5):1071–1077. doi: 10.1093/nar/25.5.1071

Intronic and exonic sequences modulate 5' splice site selection in plant nuclei.

A J McCullough 1, M A Schuler 1
PMCID: PMC146543  PMID: 9023120

Abstract

Pre-mRNA transcripts in a variety of organisms, including plants, Drosophila and Caenorhabditis elegans, contain introns which are significantly richer in adenosine and uridine residues than their flanking exons. Previous analyses using exonic and intronic replacements between two nonequivalent 5'splice sites in the 469 nt long rbcS3A intron 1 provided the first evidence indicating that, in both tobacco and Drosophila nuclei, 5'splice site selection is strongly influenced by the position of that site relative to the AU transition point between exon and intron. To differentiate between two potential models for 5'splice site recognition, we have expressed a completely different set of intronic and exonic replacement constructs containing identical 5'splice sites upstream of beta-conglycinin intron 4 (115 nt). Mutagenesis and deletion of the upstream 5'splice site demonstrate that intronic AU-rich sequences function by promoting recognition of the most upstream 5'splice site rather than by masking the downstream 5'splice site. Sequence insertions define a role for AG-rich exonic sequences in plant pre-mRNA splicing by demonstrating that an AG-rich element is capable of promoting downstream 5'splice site recognition. We conclude that AU-rich intronic sequences, AG-rich exonic sequences and the 5'splice site itself collectively define 5'intron boundaries in dicot nuclei.

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Selected References

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  1. Black D. L. Activation of c-src neuron-specific splicing by an unusual RNA element in vivo and in vitro. Cell. 1992 May 29;69(5):795–807. doi: 10.1016/0092-8674(92)90291-j. [DOI] [PubMed] [Google Scholar]
  2. Carlo T., Sterner D. A., Berget S. M. An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon. RNA. 1996 Apr;2(4):342–353. [PMC free article] [PubMed] [Google Scholar]
  3. Conrad R., Liou R. F., Blumenthal T. Conversion of a trans-spliced C. elegans gene into a conventional gene by introduction of a splice donor site. EMBO J. 1993 Mar;12(3):1249–1255. doi: 10.1002/j.1460-2075.1993.tb05766.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Conrad R., Liou R. F., Blumenthal T. Functional analysis of a C. elegans trans-splice acceptor. Nucleic Acids Res. 1993 Feb 25;21(4):913–919. doi: 10.1093/nar/21.4.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cortes J. J., Sontheimer E. J., Seiwert S. D., Steitz J. A. Mutations in the conserved loop of human U5 snRNA generate use of novel cryptic 5' splice sites in vivo. EMBO J. 1993 Dec 15;12(13):5181–5189. doi: 10.1002/j.1460-2075.1993.tb06213.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Csank C., Taylor F. M., Martindale D. W. Nuclear pre-mRNA introns: analysis and comparison of intron sequences from Tetrahymena thermophila and other eukaryotes. Nucleic Acids Res. 1990 Sep 11;18(17):5133–5141. doi: 10.1093/nar/18.17.5133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cáceres J. F., Stamm S., Helfman D. M., Krainer A. R. Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. Science. 1994 Sep 16;265(5179):1706–1709. doi: 10.1126/science.8085156. [DOI] [PubMed] [Google Scholar]
  8. Eperon I. C., Ireland D. C., Smith R. A., Mayeda A., Krainer A. R. Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF. EMBO J. 1993 Sep;12(9):3607–3617. doi: 10.1002/j.1460-2075.1993.tb06034.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ge H., Manley J. L. A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro. Cell. 1990 Jul 13;62(1):25–34. doi: 10.1016/0092-8674(90)90236-8. [DOI] [PubMed] [Google Scholar]
  10. Goodall G. J., Filipowicz W. Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants. EMBO J. 1991 Sep;10(9):2635–2644. doi: 10.1002/j.1460-2075.1991.tb07806.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goodall G. J., Filipowicz W. The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing. Cell. 1989 Aug 11;58(3):473–483. doi: 10.1016/0092-8674(89)90428-5. [DOI] [PubMed] [Google Scholar]
  12. Hanley-Bowdoin L., Elmer J. S., Rogers S. G. Transient expression of heterologous RNAs using tomato golden mosaic virus. Nucleic Acids Res. 1988 Nov 25;16(22):10511–10528. doi: 10.1093/nar/16.22.10511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huh G. S., Hynes R. O. Elements regulating an alternatively spliced exon of the rat fibronectin gene. Mol Cell Biol. 1993 Sep;13(9):5301–5314. doi: 10.1128/mcb.13.9.5301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Huh G. S., Hynes R. O. Regulation of alternative pre-mRNA splicing by a novel repeated hexanucleotide element. Genes Dev. 1994 Jul 1;8(13):1561–1574. doi: 10.1101/gad.8.13.1561. [DOI] [PubMed] [Google Scholar]
  15. Humphrey M. B., Bryan J., Cooper T. A., Berget S. M. A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon. Mol Cell Biol. 1995 Aug;15(8):3979–3988. doi: 10.1128/mcb.15.8.3979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hwang D. Y., Cohen J. B. U1 snRNA promotes the selection of nearby 5' splice sites by U6 snRNA in mammalian cells. Genes Dev. 1996 Feb 1;10(3):338–350. doi: 10.1101/gad.10.3.338. [DOI] [PubMed] [Google Scholar]
  17. Krainer A. R., Conway G. C., Kozak D. The essential pre-mRNA splicing factor SF2 influences 5' splice site selection by activating proximal sites. Cell. 1990 Jul 13;62(1):35–42. doi: 10.1016/0092-8674(90)90237-9. [DOI] [PubMed] [Google Scholar]
  18. Lavigueur A., La Branche H., Kornblihtt A. R., Chabot B. A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding. Genes Dev. 1993 Dec;7(12A):2405–2417. doi: 10.1101/gad.7.12a.2405. [DOI] [PubMed] [Google Scholar]
  19. Lazar G., Schaal T., Maniatis T., Goodman H. M. Identification of a plant serine-arginine-rich protein similar to the mammalian splicing factor SF2/ASF. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7672–7676. doi: 10.1073/pnas.92.17.7672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lin C. H., Patton J. G. Regulation of alternative 3' splice site selection by constitutive splicing factors. RNA. 1995 May;1(3):234–245. [PMC free article] [PubMed] [Google Scholar]
  21. Lopato S., Mayeda A., Krainer A. R., Barta A. Pre-mRNA splicing in plants: characterization of Ser/Arg splicing factors. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):3074–3079. doi: 10.1073/pnas.93.7.3074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lou H., Gagel R. F., Berget S. M. An intron enhancer recognized by splicing factors activates polyadenylation. Genes Dev. 1996 Jan 15;10(2):208–219. doi: 10.1101/gad.10.2.208. [DOI] [PubMed] [Google Scholar]
  23. Lou H., McCullough A. J., Schuler M. A. 3' splice site selection in dicot plant nuclei is position dependent. Mol Cell Biol. 1993 Aug;13(8):4485–4493. doi: 10.1128/mcb.13.8.4485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lou H., McCullough A. J., Schuler M. A. Expression of maize Adh1 intron mutants in tobacco nuclei. Plant J. 1993 Mar;3(3):393–403. doi: 10.1046/j.1365-313x.1993.t01-22-00999.x. [DOI] [PubMed] [Google Scholar]
  25. Mayeda A., Krainer A. R. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell. 1992 Jan 24;68(2):365–375. doi: 10.1016/0092-8674(92)90477-t. [DOI] [PubMed] [Google Scholar]
  26. McCullough A. J., Lou H., Schuler M. A. Factors affecting authentic 5' splice site selection in plant nuclei. Mol Cell Biol. 1993 Mar;13(3):1323–1331. doi: 10.1128/mcb.13.3.1323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McCullough A. J., Lou H., Schuler M. A. In vivo analysis of plant pre-mRNA splicing using an autonomously replicating vector. Nucleic Acids Res. 1991 Jun 11;19(11):3001–3009. doi: 10.1093/nar/19.11.3001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McCullough A. J., Schuler M. A. AU-rich intronic elements affect pre-mRNA 5' splice site selection in Drosophila melanogaster. Mol Cell Biol. 1993 Dec;13(12):7689–7697. doi: 10.1128/mcb.13.12.7689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nagoshi R. N., Baker B. S. Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns. Genes Dev. 1990 Jan;4(1):89–97. doi: 10.1101/gad.4.1.89. [DOI] [PubMed] [Google Scholar]
  30. Newman A. J., Norman C. U5 snRNA interacts with exon sequences at 5' and 3' splice sites. Cell. 1992 Feb 21;68(4):743–754. doi: 10.1016/0092-8674(92)90149-7. [DOI] [PubMed] [Google Scholar]
  31. Newman A., Norman C. Mutations in yeast U5 snRNA alter the specificity of 5' splice-site cleavage. Cell. 1991 Apr 5;65(1):115–123. doi: 10.1016/0092-8674(91)90413-s. [DOI] [PubMed] [Google Scholar]
  32. Ramchatesingh J., Zahler A. M., Neugebauer K. M., Roth M. B., Cooper T. A. A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer. Mol Cell Biol. 1995 Sep;15(9):4898–4907. doi: 10.1128/mcb.15.9.4898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Singh R., Valcárcel J., Green M. R. Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science. 1995 May 26;268(5214):1173–1176. doi: 10.1126/science.7761834. [DOI] [PubMed] [Google Scholar]
  34. Sun Q., Mayeda A., Hampson R. K., Krainer A. R., Rottman F. M. General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer. Genes Dev. 1993 Dec;7(12B):2598–2608. doi: 10.1101/gad.7.12b.2598. [DOI] [PubMed] [Google Scholar]
  35. Watakabe A., Tanaka K., Shimura Y. The role of exon sequences in splice site selection. Genes Dev. 1993 Mar;7(3):407–418. doi: 10.1101/gad.7.3.407. [DOI] [PubMed] [Google Scholar]
  36. Wu J., Manley J. L. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 1989 Oct;3(10):1553–1561. doi: 10.1101/gad.3.10.1553. [DOI] [PubMed] [Google Scholar]
  37. Xu R., Teng J., Cooper T. A. The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element. Mol Cell Biol. 1993 Jun;13(6):3660–3674. doi: 10.1128/mcb.13.6.3660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhuang Y., Weiner A. M. A compensatory base change in U1 snRNA suppresses a 5' splice site mutation. Cell. 1986 Sep 12;46(6):827–835. doi: 10.1016/0092-8674(86)90064-4. [DOI] [PubMed] [Google Scholar]
  39. Zhuang Y., Weiner A. M. A compensatory base change in human U2 snRNA can suppress a branch site mutation. Genes Dev. 1989 Oct;3(10):1545–1552. doi: 10.1101/gad.3.10.1545. [DOI] [PubMed] [Google Scholar]

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