Skip to main content
NCBI home page
RNA logoLink to RNA
. 2000 Mar;6(3):422–433. doi: 10.1017/s1355838200992173

Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants.

C G Simpson 1, P E Hedley 1, J A Watters 1, G P Clark 1, C McQuade 1, G C Machray 1, J W Brown 1
PMCID: PMC1369924  PMID: 10744026

Abstract

Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.

Full Text

The Full Text of this article is available as a PDF (642.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abovich N., Rosbash M. Cross-intron bridging interactions in the yeast commitment complex are conserved in mammals. Cell. 1997 May 2;89(3):403–412. doi: 10.1016/s0092-8674(00)80221-4. [DOI] [PubMed] [Google Scholar]
  2. Baynton C. E., Potthoff S. J., McCullough A. J., Schuler M. A. U-rich tracts enhance 3' splice site recognition in plant nuclei. Plant J. 1996 Oct;10(4):703–711. doi: 10.1046/j.1365-313x.1996.10040703.x. [DOI] [PubMed] [Google Scholar]
  3. Berget S. M. Exon recognition in vertebrate splicing. J Biol Chem. 1995 Feb 10;270(6):2411–2414. doi: 10.1074/jbc.270.6.2411. [DOI] [PubMed] [Google Scholar]
  4. Berglund J. A., Abovich N., Rosbash M. A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition. Genes Dev. 1998 Mar 15;12(6):858–867. doi: 10.1101/gad.12.6.858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Black D. L. Finding splice sites within a wilderness of RNA. RNA. 1995 Oct;1(8):763–771. [PMC free article] [PubMed] [Google Scholar]
  6. Bournay A. S., Hedley P. E., Maddison A., Waugh R., Machray G. C. Exon skipping induced by cold stress in a potato invertase gene transcript. Nucleic Acids Res. 1996 Jun 15;24(12):2347–2351. doi: 10.1093/nar/24.12.2347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown J. W. S., Simpson C. G. SPLICE SITE SELECTION IN PLANT PRE-mRNA SPLICING. Annu Rev Plant Physiol Plant Mol Biol. 1998 Jun;49(NaN):77–95. doi: 10.1146/annurev.arplant.49.1.77. [DOI] [PubMed] [Google Scholar]
  8. Brown J. W. Arabidopsis intron mutations and pre-mRNA splicing. Plant J. 1996 Nov;10(5):771–780. doi: 10.1046/j.1365-313x.1996.10050771.x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Cejudo F. J., Murphy G., Chinoy C., Baulcombe D. C. A gibberellin-regulated gene from wheat with sequence homology to cathepsin B of mammalian cells. Plant J. 1992 Nov;2(6):937–948. [PubMed] [Google Scholar]
  11. Chabot B., Blanchette M., Lapierre I., La Branche H. An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1. Mol Cell Biol. 1997 Apr;17(4):1776–1786. doi: 10.1128/mcb.17.4.1776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chabot B. Directing alternative splicing: cast and scenarios. Trends Genet. 1996 Nov;12(11):472–478. doi: 10.1016/0168-9525(96)10037-8. [DOI] [PubMed] [Google Scholar]
  13. Chou M. Y., Rooke N., Turck C. W., Black D. L. hnRNP H is a component of a splicing enhancer complex that activates a c-src alternative exon in neuronal cells. Mol Cell Biol. 1999 Jan;19(1):69–77. doi: 10.1128/mcb.19.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Coolidge C. J., Seely R. J., Patton J. G. Functional analysis of the polypyrimidine tract in pre-mRNA splicing. Nucleic Acids Res. 1997 Feb 15;25(4):888–896. doi: 10.1093/nar/25.4.888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cooper T. A. Muscle-specific splicing of a heterologous exon mediated by a single muscle-specific splicing enhancer from the cardiac troponin T gene. Mol Cell Biol. 1998 Aug;18(8):4519–4525. doi: 10.1128/mcb.18.8.4519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Del Gatto F., Breathnach R. Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon. Mol Cell Biol. 1995 Sep;15(9):4825–4834. doi: 10.1128/mcb.15.9.4825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dominski Z., Kole R. Selection of splice sites in pre-mRNAs with short internal exons. Mol Cell Biol. 1991 Dec;11(12):6075–6083. doi: 10.1128/mcb.11.12.6075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Domon C., Lorković Z. J., Valcárcel J., Filipowicz W. Multiple forms of the U2 small nuclear ribonucleoprotein auxiliary factor U2AF subunits expressed in higher plants. J Biol Chem. 1998 Dec 18;273(51):34603–34610. doi: 10.1074/jbc.273.51.34603. [DOI] [PubMed] [Google Scholar]
  19. Elliott K. J., Butler W. O., Dickinson C. D., Konno Y., Vedvick T. S., Fitzmaurice L., Mirkov T. E. Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening. Plant Mol Biol. 1993 Feb;21(3):515–524. doi: 10.1007/BF00028808. [DOI] [PubMed] [Google Scholar]
  20. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  21. Gallego M. E., Gattoni R., Stévenin J., Marie J., Expert-Bezançon A. The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A. EMBO J. 1997 Apr 1;16(7):1772–1784. doi: 10.1093/emboj/16.7.1772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gniadkowski M., Hemmings-Mieszczak M., Klahre U., Liu H. X., Filipowicz W. Characterization of intronic uridine-rich sequence elements acting as possible targets for nuclear proteins during pre-mRNA splicing in Nicotiana plumbaginifolia. Nucleic Acids Res. 1996 Feb 15;24(4):619–627. doi: 10.1093/nar/24.4.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Grabowski P. J. Splicing regulation in neurons: tinkering with cell-specific control. Cell. 1998 Mar 20;92(6):709–712. doi: 10.1016/s0092-8674(00)81399-9. [DOI] [PubMed] [Google Scholar]
  25. Hertel K. J., Maniatis T. Serine-arginine (SR)-rich splicing factors have an exon-independent function in pre-mRNA splicing. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2651–2655. doi: 10.1073/pnas.96.6.2651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hertel K. J., Maniatis T. The function of multisite splicing enhancers. Mol Cell. 1998 Feb;1(3):449–455. doi: 10.1016/s1097-2765(00)80045-3. [DOI] [PubMed] [Google Scholar]
  27. Hoffman B. E., Grabowski P. J. U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon. Genes Dev. 1992 Dec;6(12B):2554–2568. doi: 10.1101/gad.6.12b.2554. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Hwang D. Y., Cohen J. B. U1 small nuclear RNA-promoted exon selection requires a minimal distance between the position of U1 binding and the 3' splice site across the exon. Mol Cell Biol. 1997 Dec;17(12):7099–7107. doi: 10.1128/mcb.17.12.7099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kawamoto S. Neuron-specific alternative splicing of nonmuscle myosin II heavy chain-B pre-mRNA requires a cis-acting intron sequence. J Biol Chem. 1996 Jul 26;271(30):17613–17616. [PubMed] [Google Scholar]
  31. Ko C. H., Brendel V., Taylor R. D., Walbot V. U-richness is a defining feature of plant introns and may function as an intron recognition signal in maize. Plant Mol Biol. 1998 Mar;36(4):573–583. doi: 10.1023/a:1005932620374. [DOI] [PubMed] [Google Scholar]
  32. Kosaki A., Nelson J., Webster N. J. Identification of intron and exon sequences involved in alternative splicing of insulin receptor pre-mRNA. J Biol Chem. 1998 Apr 24;273(17):10331–10337. doi: 10.1074/jbc.273.17.10331. [DOI] [PubMed] [Google Scholar]
  33. Lim L. P., Sharp P. A. Alternative splicing of the fibronectin EIIIB exon depends on specific TGCATG repeats. Mol Cell Biol. 1998 Jul;18(7):3900–3906. doi: 10.1128/mcb.18.7.3900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Liu H. X., Filipowicz W. Mapping of branchpoint nucleotides in mutant pre-mRNAs expressed in plant cells. Plant J. 1996 Mar;9(3):381–389. doi: 10.1046/j.1365-313x.1996.09030381.x. [DOI] [PubMed] [Google Scholar]
  35. Liu H. X., Goodall G. J., Kole R., Filipowicz W. Effects of secondary structure on pre-mRNA splicing: hairpins sequestering the 5' but not the 3' splice site inhibit intron processing in Nicotiana plumbaginifolia. EMBO J. 1995 Jan 16;14(2):377–388. doi: 10.1002/j.1460-2075.1995.tb07012.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lopez A. J. Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation. Annu Rev Genet. 1998;32:279–305. doi: 10.1146/annurev.genet.32.1.279. [DOI] [PubMed] [Google Scholar]
  37. Lorenz K., Lienhard S., Sturm A. Structural organization and differential expression of carrot beta-fructofuranosidase genes: identification of a gene coding for a flower bud-specific isozyme. Plant Mol Biol. 1995 Apr;28(1):189–194. doi: 10.1007/BF00042049. [DOI] [PubMed] [Google Scholar]
  38. McCarthy E. M., Phillips J. A., 3rd Characterization of an intron splice enhancer that regulates alternative splicing of human GH pre-mRNA. Hum Mol Genet. 1998 Sep;7(9):1491–1496. doi: 10.1093/hmg/7.9.1491. [DOI] [PubMed] [Google Scholar]
  39. Mercier R. W., Gogarten J. P. A second cell wall acid invertase gene in Arabidopsis thaliana. Plant Physiol. 1995 Feb;107(2):659–660. doi: 10.1104/pp.107.2.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Min H., Turck C. W., Nikolic J. M., Black D. L. A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer. Genes Dev. 1997 Apr 15;11(8):1023–1036. doi: 10.1101/gad.11.8.1023. [DOI] [PubMed] [Google Scholar]
  41. Modafferi E. F., Black D. L. A complex intronic splicing enhancer from the c-src pre-mRNA activates inclusion of a heterologous exon. Mol Cell Biol. 1997 Nov;17(11):6537–6545. doi: 10.1128/mcb.17.11.6537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Modafferi E. F., Black D. L. Combinatorial control of a neuron-specific exon. RNA. 1999 May;5(5):687–706. doi: 10.1017/s1355838299990155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Mullen M. P., Smith C. W., Patton J. G., Nadal-Ginard B. Alpha-tropomyosin mutually exclusive exon selection: competition between branchpoint/polypyrimidine tracts determines default exon choice. Genes Dev. 1991 Apr;5(4):642–655. doi: 10.1101/gad.5.4.642. [DOI] [PubMed] [Google Scholar]
  44. Ramloch-Lorenz K., Knudsen S., Sturm A. Molecular characterization of the gene for carrot cell wall beta-fructosidase. Plant J. 1993 Sep;4(3):545–554. doi: 10.1046/j.1365-313x.1993.04030545.x. [DOI] [PubMed] [Google Scholar]
  45. Ruskin B., Zamore P. D., Green M. R. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. doi: 10.1016/0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
  46. Rutz B., Séraphin B. Transient interaction of BBP/ScSF1 and Mud2 with the splicing machinery affects the kinetics of spliceosome assembly. RNA. 1999 Jun;5(6):819–831. doi: 10.1017/s1355838299982286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ryan K. J., Cooper T. A. Muscle-specific splicing enhancers regulate inclusion of the cardiac troponin T alternative exon in embryonic skeletal muscle. Mol Cell Biol. 1996 Aug;16(8):4014–4023. doi: 10.1128/mcb.16.8.4014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schwebel-Dugué N., el Mtili N., Krivitzky M., Jean-Jacques I., Williams J. H., Thomas M., Kreis M., Lecharny A. Arabidopsis gene and cDNA encoding cell-wall invertase. Plant Physiol. 1994 Feb;104(2):809–810. doi: 10.1104/pp.104.2.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sieburth L. E., Running M. P., Meyerowitz E. M. Genetic separation of third and fourth whorl functions of AGAMOUS. Plant Cell. 1995 Aug;7(8):1249–1258. doi: 10.1105/tpc.7.8.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Simpson C. G., Clark G., Davidson D., Smith P., Brown J. W. Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency. Plant J. 1996 Mar;9(3):369–380. doi: 10.1046/j.1365-313x.1996.09030369.x. [DOI] [PubMed] [Google Scholar]
  51. Simpson C. G., McQuade C., Lyon J., Brown J. W. Characterization of exon skipping mutants of the COP1 gene from Arabidopsis. Plant J. 1998 Jul;15(1):125–131. doi: 10.1046/j.1365-313x.1998.00184.x. [DOI] [PubMed] [Google Scholar]
  52. Simpson G. G., Filipowicz W. Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organisation of the spliceosomal machinery. Plant Mol Biol. 1996 Oct;32(1-2):1–41. doi: 10.1007/BF00039375. [DOI] [PubMed] [Google Scholar]
  53. Sirand-Pugnet P., Durosay P., Brody E., Marie J. An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken beta-tropomyosin pre-mRNA. Nucleic Acids Res. 1995 Sep 11;23(17):3501–3507. doi: 10.1093/nar/23.17.3501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Smith C. W., Chu T. T., Nadal-Ginard B. Scanning and competition between AGs are involved in 3' splice site selection in mammalian introns. Mol Cell Biol. 1993 Aug;13(8):4939–4952. doi: 10.1128/mcb.13.8.4939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sterner D. A., Berget S. M. In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit. Mol Cell Biol. 1993 May;13(5):2677–2687. doi: 10.1128/mcb.13.5.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sturm A., Chrispeels M. J. cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection. Plant Cell. 1990 Nov;2(11):1107–1119. doi: 10.1105/tpc.2.11.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tolstrup N., Rouzé P., Brunak S. A branch point consensus from Arabidopsis found by non-circular analysis allows for better prediction of acceptor sites. Nucleic Acids Res. 1997 Aug 1;25(15):3159–3163. doi: 10.1093/nar/25.15.3159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Valcárcel J., Green M. R. The SR protein family: pleiotropic functions in pre-mRNA splicing. Trends Biochem Sci. 1996 Aug;21(8):296–301. [PubMed] [Google Scholar]
  59. Wang J., Manley J. L. Regulation of pre-mRNA splicing in metazoa. Curr Opin Genet Dev. 1997 Apr;7(2):205–211. doi: 10.1016/s0959-437x(97)80130-x. [DOI] [PubMed] [Google Scholar]
  60. Wei N., Lin C. Q., Modafferi E. F., Gomes W. A., Black D. L. A unique intronic splicing enhancer controls the inclusion of the agrin Y exon. RNA. 1997 Nov;3(11):1275–1288. [PMC free article] [PubMed] [Google Scholar]
  61. Yeakley J. M., Hedjran F., Morfin J. P., Merillat N., Rosenfeld M. G., Emeson R. B. Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements. Mol Cell Biol. 1993 Oct;13(10):5999–6011. doi: 10.1128/mcb.13.10.5999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zhang L., Ashiya M., Sherman T. G., Grabowski P. J. Essential nucleotides direct neuron-specific splicing of gamma 2 pre-mRNA. RNA. 1996 Jul;2(7):682–698. [PMC free article] [PubMed] [Google Scholar]
  63. Zuo P., Maniatis T. The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing. Genes Dev. 1996 Jun 1;10(11):1356–1368. doi: 10.1101/gad.10.11.1356. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES