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. 1996 Aug;8(8):1421–1435. doi: 10.1105/tpc.8.8.1421

Structure and expression of a plant U1 snRNP 70K gene: alternative splicing of U1 snRNP 70K pre-mRNAs produces two different transcripts.

M Golovkin 1, A S Reddy 1
PMCID: PMC161266  PMID: 8776903

Abstract

The product of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) 70K (U1-70K) gene, a U1 snRNP-specific protein, has been implicated in basic as well as alternative splicing of pre-mRNAs in animals. Here, we report the isolation of full-length cDNAs and the corresponding genomic clone encoding a U1-70K protein from a plant system. The Arabidopsis U1-70K protein is encoded by a single gene, which is located on chromosome 3. Several lines of evidence indicate that two distinct transcripts (short and long) are produced from the same gene by alternative splicing of the U1-70K pre-mRNA. The alternative splicing involves inclusion or exclusion of a region (910 bp) that we named "included intron." Two transcripts were clearly detectable in all tissues tested, and the level of the transcripts varied in different organs. The deduced amino acid (427 residues) sequence from the short transcript has strong homology to the animal U1-70K protein and contains an RNA recognition motif, a glycine hinge, and an arginine-rich region characteristic of the animal U1-70K protein. The long transcript has an in-frame translational termination codon within the 910-bp included intron, resulting in a truncated protein containing only 204 amino acids. The protein encoded by the short transcript is recognized by U1 RNP-specific monoclonal antibodies and binds specifically to the Arabidopsis U1 snRNA, whereas the protein from the long transcript does not. In addition, multiple polyadenylation sites were observed in the 3' untranslated region. These results suggest a complex post-transcriptional regulation of Arabidopsis U1-70K gene expression.

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Selected References

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  1. Andersen J., Zieve G. W. Assembly and intracellular transport of snRNP particles. Bioessays. 1991 Feb;13(2):57–64. doi: 10.1002/bies.950130203. [DOI] [PubMed] [Google Scholar]
  2. Bandziulis R. J., Swanson M. S., Dreyfuss G. RNA-binding proteins as developmental regulators. Genes Dev. 1989 Apr;3(4):431–437. doi: 10.1101/gad.3.4.431. [DOI] [PubMed] [Google Scholar]
  3. Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
  4. Boggs R. T., Gregor P., Idriss S., Belote J. M., McKeown M. Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene. Cell. 1987 Aug 28;50(5):739–747. doi: 10.1016/0092-8674(87)90332-1. [DOI] [PubMed] [Google Scholar]
  5. Brown J. W., Feix G., Frendewey D. Accurate in vitro splicing of two pre-mRNA plant introns in a HeLa cell nuclear extract. EMBO J. 1986 Nov;5(11):2749–2758. doi: 10.1002/j.1460-2075.1986.tb04563.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cavener D. R., Ray S. C. Eukaryotic start and stop translation sites. Nucleic Acids Res. 1991 Jun 25;19(12):3185–3192. doi: 10.1093/nar/19.12.3185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Day I. S., Reddy A. S., Golovkin M. Isolation of a new mitotic-like cyclin from Arabidopsis: complementation of a yeast cyclin mutant with a plant cyclin. Plant Mol Biol. 1996 Feb;30(3):565–575. doi: 10.1007/BF00049332. [DOI] [PubMed] [Google Scholar]
  8. Dietrich M. A., Prenger J. P., Guilfoyle T. J. Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean. Plant Mol Biol. 1990 Aug;15(2):207–223. doi: 10.1007/BF00036908. [DOI] [PubMed] [Google Scholar]
  9. Egeland D. B., Sturtevant A. P., Schuler M. A. Molecular analysis of dicot and monocot small nuclear RNA populations. Plant Cell. 1989 Jun;1(6):633–643. doi: 10.1105/tpc.1.6.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Etzerodt M., Vignali R., Ciliberto G., Scherly D., Mattaj I. W., Philipson L. Structure and expression of a Xenopus gene encoding an snRNP protein (U1 70K). EMBO J. 1988 Dec 20;7(13):4311–4321. doi: 10.1002/j.1460-2075.1988.tb03330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Favre D. Improved phenol-based method for the isolation of DNA fragments from low melting temperature agarose gels. Biotechniques. 1992 Jul;13(1):22, 25-26. [PubMed] [Google Scholar]
  12. Fu X. D., Maniatis T. Isolation of a complementary DNA that encodes the mammalian splicing factor SC35. Science. 1992 Apr 24;256(5056):535–538. doi: 10.1126/science.1373910. [DOI] [PubMed] [Google Scholar]
  13. Ge H., Zuo P., Manley J. L. Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators. Cell. 1991 Jul 26;66(2):373–382. doi: 10.1016/0092-8674(91)90626-a. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Goralski T. J., Edström J. E., Baker B. S. The sex determination locus transformer-2 of Drosophila encodes a polypeptide with similarity to RNA binding proteins. Cell. 1989 Mar 24;56(6):1011–1018. doi: 10.1016/0092-8674(89)90634-x. [DOI] [PubMed] [Google Scholar]
  17. Green M. R. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. doi: 10.1146/annurev.cb.07.110191.003015. [DOI] [PubMed] [Google Scholar]
  18. Grotewold E., Athma P., Peterson T. Alternatively spliced products of the maize P gene encode proteins with homology to the DNA-binding domain of myb-like transcription factors. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4587–4591. doi: 10.1073/pnas.88.11.4587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. doi: 10.1126/science.1853200. [DOI] [PubMed] [Google Scholar]
  20. Görlach J., Raesecke H. R., Abel G., Wehrli R., Amrhein N., Schmid J. Organ-specific differences in the ratio of alternatively spliced chorismate synthase (LeCS2) transcripts in tomato. Plant J. 1995 Sep;8(3):451–456. doi: 10.1046/j.1365-313x.1995.08030451.x. [DOI] [PubMed] [Google Scholar]
  21. Hanley B. A., Schuler M. A. Developmental expression of plant snRNAs. Nucleic Acids Res. 1991 Nov 25;19(22):6319–6325. doi: 10.1093/nar/19.22.6319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hanley B. A., Schuler M. A. cDNA cloning of U1, U2, U4 and U5 snRNA families expressed in pea nuclei. Nucleic Acids Res. 1991 Apr 25;19(8):1861–1869. doi: 10.1093/nar/19.8.1861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Heinrichs V., Bach M., Winkelmann G., Lührmann R. U1-specific protein C needed for efficient complex formation of U1 snRNP with a 5' splice site. Science. 1990 Jan 5;247(4938):69–72. doi: 10.1126/science.2136774. [DOI] [PubMed] [Google Scholar]
  24. Hilleren P. J., Kao H. Y., Siliciano P. G. The amino-terminal domain of yeast U1-70K is necessary and sufficient for function. Mol Cell Biol. 1995 Nov;15(11):6341–6350. doi: 10.1128/mcb.15.11.6341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hirose T., Sugita M., Sugiura M. cDNA structure, expression and nucleic acid-binding properties of three RNA-binding proteins in tobacco: occurrence of tissue-specific alternative splicing. Nucleic Acids Res. 1993 Aug 25;21(17):3981–3987. doi: 10.1093/nar/21.17.3981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hornig H., Fischer U., Costas M., Rauh A., Lührmann R. Analysis of genomic clones of the murine U1RNA-associated 70-kDa protein reveals a high evolutionary conservation of the protein between human and mouse. Eur J Biochem. 1989 Jun 1;182(1):45–50. doi: 10.1111/j.1432-1033.1989.tb14798.x. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Kopriva S., Cossu R., Bauwe H. Alternative splicing results in two different transcripts for H-protein of the glycine cleavage system in the C4 species Flaveria trinervia. Plant J. 1995 Sep;8(3):435–441. doi: 10.1046/j.1365-313x.1995.08030435.x. [DOI] [PubMed] [Google Scholar]
  29. Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. doi: 10.1083/jcb.115.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Krainer A. R., Conway G. C., Kozak D. Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev. 1990 Jul;4(7):1158–1171. doi: 10.1101/gad.4.7.1158. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Kuo H. C., Nasim F. H., Grabowski P. J. Control of alternative splicing by the differential binding of U1 small nuclear ribonucleoprotein particle. Science. 1991 Mar 1;251(4997):1045–1050. doi: 10.1126/science.1825520. [DOI] [PubMed] [Google Scholar]
  33. Laski F. A., Rio D. C., Rubin G. M. Tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing. Cell. 1986 Jan 17;44(1):7–19. doi: 10.1016/0092-8674(86)90480-0. [DOI] [PubMed] [Google Scholar]
  34. Luehrsen K. R., Taha S., Walbot V. Nuclear pre-mRNA processing in higher plants. Prog Nucleic Acid Res Mol Biol. 1994;47:149–193. doi: 10.1016/s0079-6603(08)60252-4. [DOI] [PubMed] [Google Scholar]
  35. Luehrsen K. R., Walbot V. Addition of A- and U-rich sequence increases the splicing efficiency of a deleted form of a maize intron. Plant Mol Biol. 1994 Feb;24(3):449–463. doi: 10.1007/BF00024113. [DOI] [PubMed] [Google Scholar]
  36. Luehrsen K. R., Walbot V. Intron creation and polyadenylation in maize are directed by AU-rich RNA. Genes Dev. 1994 May 1;8(9):1117–1130. doi: 10.1101/gad.8.9.1117. [DOI] [PubMed] [Google Scholar]
  37. Lührmann R., Kastner B., Bach M. Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim Biophys Acta. 1990 Nov 30;1087(3):265–292. doi: 10.1016/0167-4781(90)90001-i. [DOI] [PubMed] [Google Scholar]
  38. Mount S. M., Pettersson I., Hinterberger M., Karmas A., Steitz J. A. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell. 1983 Jun;33(2):509–518. doi: 10.1016/0092-8674(83)90432-4. [DOI] [PubMed] [Google Scholar]
  39. Musci M. A., Egeland D. B., Schuler M. A. Molecular comparison of monocot and dicot U1 and U2 snRNAs. Plant J. 1992 Jul;2(4):589–599. doi: 10.1046/j.1365-313x.1992.t01-28-00999.x. [DOI] [PubMed] [Google Scholar]
  40. Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
  41. Query C. C., Bentley R. C., Keene J. D. A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell. 1989 Apr 7;57(1):89–101. doi: 10.1016/0092-8674(89)90175-x. [DOI] [PubMed] [Google Scholar]
  42. Query C. C., Keene J. D. A human autoimmune protein associated with U1 RNA contains a region of homology that is cross-reactive with retroviral p30gag antigen. Cell. 1987 Oct 23;51(2):211–220. doi: 10.1016/0092-8674(87)90148-6. [DOI] [PubMed] [Google Scholar]
  43. Reddy A. S., Safadi F., Beyette J. R., Mykles D. L. Calcium-dependent proteinase activity in root cultures of Arabidopsis. Biochem Biophys Res Commun. 1994 Mar 30;199(3):1089–1095. doi: 10.1006/bbrc.1994.1342. [DOI] [PubMed] [Google Scholar]
  44. Reddy A. S., Safadi F., Narasimhulu S. B., Golovkin M., Hu X. A novel plant calmodulin-binding protein with a kinesin heavy chain motor domain. J Biol Chem. 1996 Mar 22;271(12):7052–7060. doi: 10.1074/jbc.271.12.7052. [DOI] [PubMed] [Google Scholar]
  45. Romac J. M., Graff D. H., Keene J. D. The U1 small nuclear ribonucleoprotein (snRNP) 70K protein is transported independently of U1 snRNP particles via a nuclear localization signal in the RNA-binding domain. Mol Cell Biol. 1994 Jul;14(7):4662–4670. doi: 10.1128/mcb.14.7.4662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Romac J. M., Keene J. D. Overexpression of the arginine-rich carboxy-terminal region of U1 snRNP 70K inhibits both splicing and nucleocytoplasmic transport of mRNA. Genes Dev. 1995 Jun 1;9(11):1400–1410. doi: 10.1101/gad.9.11.1400. [DOI] [PubMed] [Google Scholar]
  47. Rosbash M., Séraphin B. Who's on first? The U1 snRNP-5' splice site interaction and splicing. Trends Biochem Sci. 1991 May;16(5):187–190. doi: 10.1016/0968-0004(91)90073-5. [DOI] [PubMed] [Google Scholar]
  48. Schwarzbauer J. E., Patel R. S., Fonda D., Hynes R. O. Multiple sites of alternative splicing of the rat fibronectin gene transcript. EMBO J. 1987 Sep;6(9):2573–2580. doi: 10.1002/j.1460-2075.1987.tb02547.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sharp P. A. Split genes and RNA splicing. Cell. 1994 Jun 17;77(6):805–815. doi: 10.1016/0092-8674(94)90130-9. [DOI] [PubMed] [Google Scholar]
  50. Smith C. W., Patton J. G., Nadal-Ginard B. Alternative splicing in the control of gene expression. Annu Rev Genet. 1989;23:527–577. doi: 10.1146/annurev.ge.23.120189.002523. [DOI] [PubMed] [Google Scholar]
  51. Smith V., Barrell B. G. Cloning of a yeast U1 snRNP 70K protein homologue: functional conservation of an RNA-binding domain between humans and yeast. EMBO J. 1991 Sep;10(9):2627–2634. doi: 10.1002/j.1460-2075.1991.tb07805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Spritz R. A., Strunk K., Surowy C. S., Hoch S. O., Barton D. E., Francke U. The human U1-70K snRNP protein: cDNA cloning, chromosomal localization, expression, alternative splicing and RNA-binding. Nucleic Acids Res. 1987 Dec 23;15(24):10373–10391. doi: 10.1093/nar/15.24.10373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tamaoki M., Tsugawa H., Minami E., Kayano T., Yamamoto N., Kano-Murakami Y., Matsuoka M. Alternative RNA products from a rice homeobox gene. Plant J. 1995 Jun;7(6):927–938. doi: 10.1046/j.1365-313x.1995.07060927.x. [DOI] [PubMed] [Google Scholar]
  54. Tazi J., Kornstädt U., Rossi F., Jeanteur P., Cathala G., Brunel C., Lührmann R. Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing. Nature. 1993 May 20;363(6426):283–286. doi: 10.1038/363283a0. [DOI] [PubMed] [Google Scholar]
  55. Tian M., Maniatis T. Positive control of pre-mRNA splicing in vitro. Science. 1992 Apr 10;256(5054):237–240. doi: 10.1126/science.1566072. [DOI] [PubMed] [Google Scholar]
  56. Vaux P., Guerineau F., Waugh R., Brown J. W. Characterization and expression of U1snRNA genes from potato. Plant Mol Biol. 1992 Sep;19(6):959–971. doi: 10.1007/BF00040528. [DOI] [PubMed] [Google Scholar]
  57. Werneke J. M., Chatfield J. M., Ogren W. L. Alternative mRNA splicing generates the two ribulosebisphosphate carboxylase/oxygenase activase polypeptides in spinach and Arabidopsis. Plant Cell. 1989 Aug;1(8):815–825. doi: 10.1105/tpc.1.8.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wiebauer K., Herrero J. J., Filipowicz W. Nuclear pre-mRNA processing in plants: distinct modes of 3'-splice-site selection in plants and animals. Mol Cell Biol. 1988 May;8(5):2042–2051. doi: 10.1128/mcb.8.5.2042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Woppmann A., Will C. L., Kornstädt U., Zuo P., Manley J. L., Lührmann R. Identification of an snRNP-associated kinase activity that phosphorylates arginine/serine rich domains typical of splicing factors. Nucleic Acids Res. 1993 Jun 25;21(12):2815–2822. doi: 10.1093/nar/21.12.2815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wu J. Y., Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. doi: 10.1016/0092-8674(93)90316-i. [DOI] [PubMed] [Google Scholar]
  61. Wu L., Ueda T., Messing J. The formation of mRNA 3'-ends in plants. Plant J. 1995 Sep;8(3):323–329. doi: 10.1046/j.1365-313x.1995.08030323.x. [DOI] [PubMed] [Google Scholar]
  62. Yuo C. Y., Weiner A. M. Genetic analysis of the role of human U1 snRNA in mRNA splicing: I. Effect of mutations in the highly conserved stem-loop I of U1. Genes Dev. 1989 May;3(5):697–707. doi: 10.1101/gad.3.5.697. [DOI] [PubMed] [Google Scholar]
  63. Zamore P. D., Patton J. G., Green M. R. Cloning and domain structure of the mammalian splicing factor U2AF. Nature. 1992 Feb 13;355(6361):609–614. doi: 10.1038/355609a0. [DOI] [PubMed] [Google Scholar]
  64. Zhang M., Zamore P. D., Carmo-Fonseca M., Lamond A. I., Green M. R. Cloning and intracellular localization of the U2 small nuclear ribonucleoprotein auxiliary factor small subunit. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8769–8773. doi: 10.1073/pnas.89.18.8769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. van Santen V. L., Spritz R. A. Splicing of plant pre-mRNAs in animal systems and vice versa. Gene. 1987;56(2-3):253–265. doi: 10.1016/0378-1119(87)90142-9. [DOI] [PubMed] [Google Scholar]

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