Skip to main content
PMC home page
RNA logoLink to RNA
. 2000 Nov;6(11):1610–1624. doi: 10.1017/s1355838200001163

RBP45 and RBP47, two oligouridylate-specific hnRNP-like proteins interacting with poly(A)+ RNA in nuclei of plant cells.

Z J Lorković 1, D A Wieczorek Kirk 1, U Klahre 1, M Hemmings-Mieszczak 1, W Filipowicz 1
PMCID: PMC1370030  PMID: 11105760

Abstract

Introns in plant nuclear pre-mRNAs are highly enriched in U or U + A residues and this property is essential for efficient splicing. Moreover, 3'-untranslated regions (3'-UTRs) in plant pre-mRNAs are generally UA-rich and contain sequences that are important for the polyadenylation reaction. Here, we characterize two structurally related RNA-binding proteins (RBPs) from Nicotiana plumbaginifolia, referred to as RBP45 and RBP47, having specificity for oligouridylates. Both proteins contain three RBD-type RNA-binding domains and a glutamine-rich N-terminus, and share similarity with Nam8p, a protein associated with U1 snRNP in the yeast Saccharomyces cerevisiae. Deletion analysis of RBP45 and RBP47 indicated that the presence of at least two RBD are required for interaction with RNA and that domains other than RBD do not significantly contribute to binding. mRNAs for RBP45 and RBP47 and mRNAs encoding six related proteins in Arabidopsis thaliana are constitutively expressed in different plant organs. Indirect immunofluorescence and fractionation of cell extracts showed that RBP45 and RBP47 are localized in the nucleus. In vivo UV crosslinking experiments demonstrated their association with the nuclear poly(A)+ RNA. In contrast to UBP1, another oligouridylate-binding nuclear three-RBD protein of N. plumbaginifolia (Lambermon et al., EMBO J, 2000, 19:1638-1649), RBP45 and RBP47 do not stimulate mRNA splicing and accumulation when transiently overexpressed in protoplasts. Properties of RBP45 and RBP47 suggest they represent hnRNP-proteins participating in still undefined steps of pre-mRNA maturation in plant cell nuclei.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Anderson J. T., Paddy M. R., Swanson M. S. PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Oct;13(10):6102–6113. doi: 10.1128/mcb.13.10.6102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Antic D., Keene J. D. Embryonic lethal abnormal visual RNA-binding proteins involved in growth, differentiation, and posttranscriptional gene expression. Am J Hum Genet. 1997 Aug;61(2):273–278. doi: 10.1086/514866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beck A. R., Medley Q. G., O'Brien S., Anderson P., Streuli M. Structure, tissue distribution and genomic organization of the murine RRM-type RNA binding proteins TIA-1 and TIAR. Nucleic Acids Res. 1996 Oct 1;24(19):3829–3835. doi: 10.1093/nar/24.19.3829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beck A. R., Miller I. J., Anderson P., Streuli M. RNA-binding protein TIAR is essential for primordial germ cell development. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2331–2336. doi: 10.1073/pnas.95.5.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beven A. F., Simpson G. G., Brown J. W., Shaw P. J. The organization of spliceosomal components in the nuclei of higher plants. J Cell Sci. 1995 Feb;108(Pt 2):509–518. doi: 10.1242/jcs.108.2.509. [DOI] [PubMed] [Google Scholar]
  6. Blencowe B. J. Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem Sci. 2000 Mar;25(3):106–110. doi: 10.1016/s0968-0004(00)01549-8. [DOI] [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. Calado A., Kutay U., Kühn U., Wahle E., Carmo-Fonseca M. Deciphering the cellular pathway for transport of poly(A)-binding protein II. RNA. 2000 Feb;6(2):245–256. doi: 10.1017/s1355838200991908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Colgan D. F., Manley J. L. Mechanism and regulation of mRNA polyadenylation. Genes Dev. 1997 Nov 1;11(21):2755–2766. doi: 10.1101/gad.11.21.2755. [DOI] [PubMed] [Google Scholar]
  10. Dember L. M., Kim N. D., Liu K. Q., Anderson P. Individual RNA recognition motifs of TIA-1 and TIAR have different RNA binding specificities. J Biol Chem. 1996 Feb 2;271(5):2783–2788. doi: 10.1074/jbc.271.5.2783. [DOI] [PubMed] [Google Scholar]
  11. Egoavil C., Marton H. A., Baynton C. E., McCullough A. J., Schuler M. A. Structural analysis of elements contributing to 5' splice site selection in plant pre-mRNA transcripts. Plant J. 1997 Nov;12(5):971–980. doi: 10.1046/j.1365-313x.1997.12050971.x. [DOI] [PubMed] [Google Scholar]
  12. Escher D., Bodmer-Glavas M., Barberis A., Schaffner W. Conservation of glutamine-rich transactivation function between yeast and humans. Mol Cell Biol. 2000 Apr;20(8):2774–2782. doi: 10.1128/mcb.20.8.2774-2782.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fan X. C., Steitz J. A. Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs. EMBO J. 1998 Jun 15;17(12):3448–3460. doi: 10.1093/emboj/17.12.3448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ford L. P., Watson J., Keene J. D., Wilusz J. ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system. Genes Dev. 1999 Jan 15;13(2):188–201. doi: 10.1101/gad.13.2.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fortes P., Bilbao-Cortés D., Fornerod M., Rigaut G., Raymond W., Séraphin B., Mattaj I. W. Luc7p, a novel yeast U1 snRNP protein with a role in 5' splice site recognition. Genes Dev. 1999 Sep 15;13(18):2425–2438. doi: 10.1101/gad.13.18.2425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gamberi C., Izaurralde E., Beisel C., Mattaj I. W. Interaction between the human nuclear cap-binding protein complex and hnRNP F. Mol Cell Biol. 1997 May;17(5):2587–2597. doi: 10.1128/mcb.17.5.2587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Genschik P., Hall J., Filipowicz W. Cloning and characterization of the Arabidopsis cyclic phosphodiesterase which hydrolyzes ADP-ribose 1'',2''-cyclic phosphate and nucleoside 2',3'-cyclic phosphates. J Biol Chem. 1997 May 16;272(20):13211–13219. doi: 10.1074/jbc.272.20.13211. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Good P. J. A conserved family of elav-like genes in vertebrates. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4557–4561. doi: 10.1073/pnas.92.10.4557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Goodall G. J., Wiebauer K., Filipowicz W. Analysis of pre-mRNA processing in transfected plant protoplasts. Methods Enzymol. 1990;181:148–161. doi: 10.1016/0076-6879(90)81117-d. [DOI] [PubMed] [Google Scholar]
  22. Gottschalk A., Tang J., Puig O., Salgado J., Neubauer G., Colot H. V., Mann M., Séraphin B., Rosbash M., Lührmann R. A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins. RNA. 1998 Apr;4(4):374–393. [PMC free article] [PubMed] [Google Scholar]
  23. Gueydan C., Droogmans L., Chalon P., Huez G., Caput D., Kruys V. Identification of TIAR as a protein binding to the translational regulatory AU-rich element of tumor necrosis factor alpha mRNA. J Biol Chem. 1999 Jan 22;274(4):2322–2326. doi: 10.1074/jbc.274.4.2322. [DOI] [PubMed] [Google Scholar]
  24. Hanano S., Sugita M., Sugiura M. Isolation of a novel RNA-binding protein and its association with a large ribonucleoprotein particle present in the nucleoplasm of tobacco cells. Plant Mol Biol. 1996 Apr;31(1):57–68. doi: 10.1007/BF00020606. [DOI] [PubMed] [Google Scholar]
  25. Izaurralde E., Adam S. Transport of macromolecules between the nucleus and the cytoplasm. RNA. 1998 Apr;4(4):351–364. [PMC free article] [PubMed] [Google Scholar]
  26. Jack T., Brockman L. L., Meyerowitz E. M. The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell. 1992 Feb 21;68(4):683–697. doi: 10.1016/0092-8674(92)90144-2. [DOI] [PubMed] [Google Scholar]
  27. Keller W., Minvielle-Sebastia L. A comparison of mammalian and yeast pre-mRNA 3'-end processing. Curr Opin Cell Biol. 1997 Jun;9(3):329–336. doi: 10.1016/s0955-0674(97)80004-x. [DOI] [PubMed] [Google Scholar]
  28. Kessler M. M., Henry M. F., Shen E., Zhao J., Gross S., Silver P. A., Moore C. L. Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3'-end formation in yeast. Genes Dev. 1997 Oct 1;11(19):2545–2556. doi: 10.1101/gad.11.19.2545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kim Y. J., Baker B. S. The Drosophila gene rbp9 encodes a protein that is a member of a conserved group of putative RNA binding proteins that are nervous system-specific in both flies and humans. J Neurosci. 1993 Mar;13(3):1045–1056. doi: 10.1523/JNEUROSCI.13-03-01045.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. King P. H., Levine T. D., Fremeau R. T., Jr, Keene J. D. Mammalian homologs of Drosophila ELAV localized to a neuronal subset can bind in vitro to the 3' UTR of mRNA encoding the Id transcriptional repressor. J Neurosci. 1994 Apr;14(4):1943–1952. doi: 10.1523/JNEUROSCI.14-04-01943.1994. [DOI] [PMC free article] [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. Koushika S. P., Lisbin M. J., White K. ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform. Curr Biol. 1996 Dec 1;6(12):1634–1641. doi: 10.1016/s0960-9822(02)70787-2. [DOI] [PubMed] [Google Scholar]
  33. Krecic A. M., Swanson M. S. hnRNP complexes: composition, structure, and function. Curr Opin Cell Biol. 1999 Jun;11(3):363–371. doi: 10.1016/S0955-0674(99)80051-9. [DOI] [PubMed] [Google Scholar]
  34. Krämer A. The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu Rev Biochem. 1996;65:367–409. doi: 10.1146/annurev.bi.65.070196.002055. [DOI] [PubMed] [Google Scholar]
  35. Lambermon M. H., Simpson G. G., Wieczorek Kirk D. A., Hemmings-Mieszczak M., Klahre U., Filipowicz W. UBP1, a novel hnRNP-like protein that functions at multiple steps of higher plant nuclear pre-mRNA maturation. EMBO J. 2000 Apr 3;19(7):1638–1649. doi: 10.1093/emboj/19.7.1638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lambermon M. H., Simpson G. G., Wieczorek Kirk D. A., Hemmings-Mieszczak M., Klahre U., Filipowicz W. UBP1, a novel hnRNP-like protein that functions at multiple steps of higher plant nuclear pre-mRNA maturation. EMBO J. 2000 Apr 3;19(7):1638–1649. doi: 10.1093/emboj/19.7.1638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lane R. D., Crissman R. S., Ginn S. High efficiency fusion procedure for producing monoclonal antibodies against weak immunogens. Methods Enzymol. 1986;121:183–192. doi: 10.1016/0076-6879(86)21017-4. [DOI] [PubMed] [Google Scholar]
  38. Levy N. S., Chung S., Furneaux H., Levy A. P. Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR. J Biol Chem. 1998 Mar 13;273(11):6417–6423. doi: 10.1074/jbc.273.11.6417. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Lorković Z. J., Wieczorek Kirk D. A., Lambermon M. H., Filipowicz W. Pre-mRNA splicing in higher plants. Trends Plant Sci. 2000 Apr;5(4):160–167. doi: 10.1016/s1360-1385(00)01595-8. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Ma W. J., Cheng S., Campbell C., Wright A., Furneaux H. Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein. J Biol Chem. 1996 Apr 5;271(14):8144–8151. doi: 10.1074/jbc.271.14.8144. [DOI] [PubMed] [Google Scholar]
  44. Matunis M. J., Matunis E. L., Dreyfuss G. PUB1: a major yeast poly(A)+ RNA-binding protein. Mol Cell Biol. 1993 Oct;13(10):6114–6123. doi: 10.1128/mcb.13.10.6114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Michael W. M., Siomi H., Choi M., Piñol-Roma S., Nakielny S., Liu Q., Dreyfuss G. Signal sequences that target nuclear import and nuclear export of pre-mRNA-binding proteins. Cold Spring Harb Symp Quant Biol. 1995;60:663–668. doi: 10.1101/sqb.1995.060.01.071. [DOI] [PubMed] [Google Scholar]
  47. Mieszczak M., Klahre U., Levy J. H., Goodall G. J., Filipowicz W. Multiple plant RNA binding proteins identified by PCR: expression of cDNAs encoding RNA binding proteins targeted to chloroplasts in Nicotiana plumbaginifolia. Mol Gen Genet. 1992 Sep;234(3):390–400. doi: 10.1007/BF00538698. [DOI] [PubMed] [Google Scholar]
  48. Minvielle-Sebastia L., Beyer K., Krecic A. M., Hector R. E., Swanson M. S., Keller W. Control of cleavage site selection during mRNA 3' end formation by a yeast hnRNP. EMBO J. 1998 Dec 15;17(24):7454–7468. doi: 10.1093/emboj/17.24.7454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Moriguchi K., Sugita M., Sugiura M. Structure and subcellular localization of a small RNA-binding protein from tobacco. Plant J. 1997 Jul;12(1):215–221. doi: 10.1046/j.1365-313x.1997.12010215.x. [DOI] [PubMed] [Google Scholar]
  50. Nakielny S., Fischer U., Michael W. M., Dreyfuss G. RNA transport. Annu Rev Neurosci. 1997;20:269–301. doi: 10.1146/annurev.neuro.20.1.269. [DOI] [PubMed] [Google Scholar]
  51. Peng S. S., Chen C. Y., Xu N., Shyu A. B. RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein. EMBO J. 1998 Jun 15;17(12):3461–3470. doi: 10.1093/emboj/17.12.3461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Puig O., Gottschalk A., Fabrizio P., Séraphin B. Interaction of the U1 snRNP with nonconserved intronic sequences affects 5' splice site selection. Genes Dev. 1999 Mar 1;13(5):569–580. doi: 10.1101/gad.13.5.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Robinow S., Campos A. R., Yao K. M., White K. The elav gene product of Drosophila, required in neurons, has three RNP consensus motifs. Science. 1988 Dec 16;242(4885):1570–1572. doi: 10.1126/science.3144044. [DOI] [PubMed] [Google Scholar]
  54. Rothnie H. M. Plant mRNA 3'-end formation. Plant Mol Biol. 1996 Oct;32(1-2):43–61. doi: 10.1007/BF00039376. [DOI] [PubMed] [Google Scholar]
  55. Sablowski R. W., Meyerowitz E. M. Temperature-sensitive splicing in the floral homeotic mutant apetala3-1. Plant Cell. 1998 Sep;10(9):1453–1463. doi: 10.1105/tpc.10.9.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. 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]
  57. Siomi H., Dreyfuss G. RNA-binding proteins as regulators of gene expression. Curr Opin Genet Dev. 1997 Jun;7(3):345–353. doi: 10.1016/s0959-437x(97)80148-7. [DOI] [PubMed] [Google Scholar]
  58. Spitz M. "Single-shot" intrasplenic immunization for the production of monoclonal antibodies. Methods Enzymol. 1986;121:33–41. doi: 10.1016/0076-6879(86)21006-x. [DOI] [PubMed] [Google Scholar]
  59. Szabo A., Dalmau J., Manley G., Rosenfeld M., Wong E., Henson J., Posner J. B., Furneaux H. M. HuD, a paraneoplastic encephalomyelitis antigen, contains RNA-binding domains and is homologous to Elav and Sex-lethal. Cell. 1991 Oct 18;67(2):325–333. doi: 10.1016/0092-8674(91)90184-z. [DOI] [PubMed] [Google Scholar]
  60. Séguin A., Laible G., Leyva A., Dixon R. A., Lamb C. J. Characterization of a gene encoding a DNA-binding protein that interacts in vitro with vascular specific cis elements of the phenylalanine ammonia-lyase promoter. Plant Mol Biol. 1997 Oct;35(3):281–291. doi: 10.1023/a:1005853404242. [DOI] [PubMed] [Google Scholar]
  61. 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]
  62. 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]
  63. Wahle E., Rüegsegger U. 3'-End processing of pre-mRNA in eukaryotes. FEMS Microbiol Rev. 1999 Jun;23(3):277–295. doi: 10.1111/j.1574-6976.1999.tb00400.x. [DOI] [PubMed] [Google Scholar]
  64. Weighardt F., Biamonti G., Riva S. The roles of heterogeneous nuclear ribonucleoproteins (hnRNP) in RNA metabolism. Bioessays. 1996 Sep;18(9):747–756. doi: 10.1002/bies.950180910. [DOI] [PubMed] [Google Scholar]
  65. Wilson S. M., Datar K. V., Paddy M. R., Swedlow J. R., Swanson M. S. Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces cerevisiae. J Cell Biol. 1994 Dec;127(5):1173–1184. doi: 10.1083/jcb.127.5.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yi Y., Jack T. An intragenic suppressor of the Arabidopsis floral organ identity mutant apetala3-1 functions by suppressing defects in splicing. Plant Cell. 1998 Sep;10(9):1465–1477. doi: 10.1105/tpc.10.9.1465. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES