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. 2001 Jun;7(6):859–874. doi: 10.1017/s1355838201002084

A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA.

M L Hastings 1, C M Wilson 1, S H Munroe 1
PMCID: PMC1370135  PMID: 11421362

Abstract

The mammalian thyroid hormone receptor gene c-erbAalpha gives rise to two mRNAs that code for distinct isoforms, TRalpha1 and TRalpha2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRalpha1-specific polyadenylation site or TRalpha2-specific 5' splice site. A previous investigation of TRalpha minigene expression defined a critical role for the TRalpha2 5' splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEa2, enhance splicing of TRalpha2 in vitro as well as in vivo. Although SEalpha2 is located within the intron of TRalpha2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEalpha2 functions by binding trans-acting factors in HeLa nuclear extract. Protein-RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEalpha2. SEalpha2 also includes an element resembling a 5' splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5' splice site sequence have a dramatic effect on splicing and protein binding. Thus SEa2 and its associated factors are required for splicing of TRalpha2 pre-mRNA.

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Selected References

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  1. 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]
  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. Carstens R. P., McKeehan W. L., Garcia-Blanco M. A. An intronic sequence element mediates both activation and repression of rat fibroblast growth factor receptor 2 pre-mRNA splicing. Mol Cell Biol. 1998 Apr;18(4):2205–2217. doi: 10.1128/mcb.18.4.2205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen C. D., Kobayashi R., Helfman D. M. Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene. Genes Dev. 1999 Mar 1;13(5):593–606. doi: 10.1101/gad.13.5.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Fogel B. L., McNally M. T. A cellular protein, hnRNP H, binds to the negative regulator of splicing element from Rous sarcoma virus. J Biol Chem. 2000 Oct 13;275(41):32371–32378. doi: 10.1074/jbc.M005000200. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Gontarek R. R., McNally M. T., Beemon K. Mutation of an RSV intronic element abolishes both U11/U12 snRNP binding and negative regulation of splicing. Genes Dev. 1993 Oct;7(10):1926–1936. doi: 10.1101/gad.7.10.1926. [DOI] [PubMed] [Google Scholar]
  10. Gontarek R. R., McNally M. T., Beemon K. Mutation of an RSV intronic element abolishes both U11/U12 snRNP binding and negative regulation of splicing. Genes Dev. 1993 Oct;7(10):1926–1936. doi: 10.1101/gad.7.10.1926. [DOI] [PubMed] [Google Scholar]
  11. Gooding C., Roberts G. C., Moreau G., Nadal-Ginard B., Smith C. W. Smooth muscle-specific switching of alpha-tropomyosin mutually exclusive exon selection by specific inhibition of the strong default exon. EMBO J. 1994 Aug 15;13(16):3861–3872. doi: 10.1002/j.1460-2075.1994.tb06697.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Graveley B. R. Sorting out the complexity of SR protein functions. RNA. 2000 Sep;6(9):1197–1211. doi: 10.1017/s1355838200000960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hanamura A., Cáceres J. F., Mayeda A., Franza B. R., Jr, Krainer A. R. Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors. RNA. 1998 Apr;4(4):430–444. [PMC free article] [PubMed] [Google Scholar]
  14. Hastings M. L., Ingle H. A., Lazar M. A., Munroe S. H. Post-transcriptional regulation of thyroid hormone receptor expression by cis-acting sequences and a naturally occurring antisense RNA. J Biol Chem. 2000 Apr 14;275(15):11507–11513. doi: 10.1074/jbc.275.15.11507. [DOI] [PubMed] [Google Scholar]
  15. Hastings M. L., Milcarek C., Martincic K., Peterson M. L., Munroe S. H. Expression of the thyroid hormone receptor gene, erbAalpha, in B lymphocytes: alternative mRNA processing is independent of differentiation but correlates with antisense RNA levels. Nucleic Acids Res. 1997 Nov 1;25(21):4296–4300. doi: 10.1093/nar/25.21.4296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hertel K. J., Lynch K. W., Maniatis T. Common themes in the function of transcription and splicing enhancers. Curr Opin Cell Biol. 1997 Jun;9(3):350–357. doi: 10.1016/s0955-0674(97)80007-5. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Jamison S. F., Pasman Z., Wang J., Will C., Lührmann R., Manley J. L., Garcia-Blanco M. A. U1 snRNP-ASF/SF2 interaction and 5' splice site recognition: characterization of required elements. Nucleic Acids Res. 1995 Aug 25;23(16):3260–3267. doi: 10.1093/nar/23.16.3260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Koenig R. J., Lazar M. A., Hodin R. A., Brent G. A., Larsen P. R., Chin W. W., Moore D. D. Inhibition of thyroid hormone action by a non-hormone binding c-erbA protein generated by alternative mRNA splicing. Nature. 1989 Feb 16;337(6208):659–661. doi: 10.1038/337659a0. [DOI] [PubMed] [Google Scholar]
  21. Kohtz J. D., Jamison S. F., Will C. L., Zuo P., Lührmann R., Garcia-Blanco M. A., Manley J. L. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 1994 Mar 10;368(6467):119–124. doi: 10.1038/368119a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Krainer A. R., Maniatis T. Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro. Cell. 1985 Oct;42(3):725–736. doi: 10.1016/0092-8674(85)90269-7. [DOI] [PubMed] [Google Scholar]
  24. Krainer A. R., Maniatis T., Ruskin B., Green M. R. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. doi: 10.1016/0092-8674(84)90049-7. [DOI] [PubMed] [Google Scholar]
  25. Krainer A. R., Mayeda A., Kozak D., Binns G. Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators. Cell. 1991 Jul 26;66(2):383–394. doi: 10.1016/0092-8674(91)90627-b. [DOI] [PubMed] [Google Scholar]
  26. Laudet V., Begue A., Henry-Duthoit C., Joubel A., Martin P., Stehelin D., Saule S. Genomic organization of the human thyroid hormone receptor alpha (c-erbA-1) gene. Nucleic Acids Res. 1991 Mar 11;19(5):1105–1112. doi: 10.1093/nar/19.5.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lazar M. A., Hodin R. A., Chin W. W. Human carboxyl-terminal variant of alpha-type c-erbA inhibits trans-activation by thyroid hormone receptors without binding thyroid hormone. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7771–7774. doi: 10.1073/pnas.86.20.7771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lazar M. A., Hodin R. A., Darling D. S., Chin W. W. A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbA alpha transcriptional unit. Mol Cell Biol. 1989 Mar;9(3):1128–1136. doi: 10.1128/mcb.9.3.1128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lazar M. A., Hodin R. A., Darling D. S., Chin W. W. Identification of a rat c-erbA alpha-related protein which binds deoxyribonucleic acid but does not bind thyroid hormone. Mol Endocrinol. 1988 Oct;2(10):893–901. doi: 10.1210/mend-2-10-893. [DOI] [PubMed] [Google Scholar]
  30. Lazar M. A. Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev. 1993 Apr;14(2):184–193. doi: 10.1210/edrv-14-2-184. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Liu H. X., Zhang M., Krainer A. R. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev. 1998 Jul 1;12(13):1998–2012. doi: 10.1101/gad.12.13.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lou H., Neugebauer K. M., Gagel R. F., Berget S. M. Regulation of alternative polyadenylation by U1 snRNPs and SRp20. Mol Cell Biol. 1998 Sep;18(9):4977–4985. doi: 10.1128/mcb.18.9.4977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lou H., Yang Y., Cote G. J., Berget S. M., Gagel R. F. An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene. Mol Cell Biol. 1995 Dec;15(12):7135–7142. doi: 10.1128/mcb.15.12.7135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Matunis M. J., Xing J., Dreyfuss G. The hnRNP F protein: unique primary structure, nucleic acid-binding properties, and subcellular localization. Nucleic Acids Res. 1994 Mar 25;22(6):1059–1067. doi: 10.1093/nar/22.6.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. McCullough A. J., Berget S. M. An intronic splicing enhancer binds U1 snRNPs to enhance splicing and select 5' splice sites. Mol Cell Biol. 2000 Dec;20(24):9225–9235. doi: 10.1128/mcb.20.24.9225-9235.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. McCullough A. J., Berget S. M. G triplets located throughout a class of small vertebrate introns enforce intron borders and regulate splice site selection. Mol Cell Biol. 1997 Aug;17(8):4562–4571. doi: 10.1128/mcb.17.8.4562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. McNally L. M., McNally M. T. An RNA splicing enhancer-like sequence is a component of a splicing inhibitor element from Rous sarcoma virus. Mol Cell Biol. 1998 Jun;18(6):3103–3111. doi: 10.1128/mcb.18.6.3103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. McNally L. M., McNally M. T. U1 small nuclear ribonucleoprotein and splicing inhibition by the rous sarcoma virus negative regulator of splicing element. J Virol. 1999 Mar;73(3):2385–2393. doi: 10.1128/jvi.73.3.2385-2393.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Min H., Chan R. C., Black D. L. The generally expressed hnRNP F is involved in a neural-specific pre-mRNA splicing event. Genes Dev. 1995 Nov 1;9(21):2659–2671. doi: 10.1101/gad.9.21.2659. [DOI] [PubMed] [Google Scholar]
  42. Munroe S. H. Antisense RNA inhibits splicing of pre-mRNA in vitro. EMBO J. 1988 Aug;7(8):2523–2532. doi: 10.1002/j.1460-2075.1988.tb03100.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Munroe S. H., Lazar M. A. Inhibition of c-erbA mRNA splicing by a naturally occurring antisense RNA. J Biol Chem. 1991 Nov 25;266(33):22083–22086. [PubMed] [Google Scholar]
  44. Nussinov R. Conserved quartets near 5' intron junctions in primate nuclear pre-mRNA. J Theor Biol. 1988 Jul 8;133(1):73–84. doi: 10.1016/s0022-5193(88)80025-0. [DOI] [PubMed] [Google Scholar]
  45. Robberson B. L., Cote G. J., Berget S. M. Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol. 1990 Jan;10(1):84–94. doi: 10.1128/mcb.10.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schaal T. D., Maniatis T. Multiple distinct splicing enhancers in the protein-coding sequences of a constitutively spliced pre-mRNA. Mol Cell Biol. 1999 Jan;19(1):261–273. doi: 10.1128/mcb.19.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Screaton G. R., Cáceres J. F., Mayeda A., Bell M. V., Plebanski M., Jackson D. G., Bell J. I., Krainer A. R. Identification and characterization of three members of the human SR family of pre-mRNA splicing factors. EMBO J. 1995 Sep 1;14(17):4336–4349. doi: 10.1002/j.1460-2075.1995.tb00108.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Siebel C. W., Fresco L. D., Rio D. C. The mechanism of somatic inhibition of Drosophila P-element pre-mRNA splicing: multiprotein complexes at an exon pseudo-5' splice site control U1 snRNP binding. Genes Dev. 1992 Aug;6(8):1386–1401. doi: 10.1101/gad.6.8.1386. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Smith C. W., Valcárcel J. Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci. 2000 Aug;25(8):381–388. doi: 10.1016/s0968-0004(00)01604-2. [DOI] [PubMed] [Google Scholar]
  51. Staknis D., Reed R. SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex. Mol Cell Biol. 1994 Nov;14(11):7670–7682. doi: 10.1128/mcb.14.11.7670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tacke R., Manley J. L. Determinants of SR protein specificity. Curr Opin Cell Biol. 1999 Jun;11(3):358–362. doi: 10.1016/S0955-0674(99)80050-7. [DOI] [PubMed] [Google Scholar]
  53. Tanaka K., Watakabe A., Shimura Y. Polypurine sequences within a downstream exon function as a splicing enhancer. Mol Cell Biol. 1994 Feb;14(2):1347–1354. doi: 10.1128/mcb.14.2.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tian M., Maniatis T. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell. 1993 Jul 16;74(1):105–114. doi: 10.1016/0092-8674(93)90298-5. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. 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]
  57. Zahler A. M. Purification of SR protein splicing factors. Methods Mol Biol. 1999;118:419–432. doi: 10.1385/1-59259-676-2:419. [DOI] [PubMed] [Google Scholar]

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