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. 1997 Apr;17(4):2257–2265. doi: 10.1128/mcb.17.4.2257

PRH75, a new nucleus-localized member of the DEAD-box protein family from higher plants.

Z J Lorković 1, R G Herrmann 1, R Oelmüller 1
PMCID: PMC232075  PMID: 9121476

Abstract

The putative RNA helicases of the DEAD-box protein family are involved in pre-mRNA splicing, rRNA maturation, ribosome assembly, and translation. Members of this protein family have been identified in organisms from Escherichia coli to humans, but except for the translation initiation factor 4A, there have been no reports on the characterization of other DEAD-box proteins from plants. Here we report on a novel member of the DEAD-box protein family, the plant RNA helicase 75 (PRH75). PRH75 is localized in the nucleus and contains two domains for RNA binding. One is located at the C terminus and is similar to RGG RNA-binding domains of nucleus-localized RNA-binding proteins. The other one is located between amino acids 308 and 622, a region containing the conserved motif VI characteristic of DEAD-box proteins and known as the RNA-binding site of eIF-4A. The N-terminal 81 amino acids are sufficient for nuclear targeting of the protein. Northern and Western blot analyses show that PRH75 is mainly expressed in young and rapidly developing tissues. The purified recombinant PRH75 has a weak ATPase activity which is barely stimulated by RNA ligands. The fractionation of spinach whole-cell extracts by glycerol gradient centrifugation and gel filtration on a Superdex 200 column shows that the protein exists in a complex of about 500 kDa. Possible biological functions of PRH75 as well as structure-function relationships in the context of its modular primary structure are discussed.

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Selected References

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  1. Abramson R. D., Dever T. E., Lawson T. G., Ray B. K., Thach R. E., Merrick W. C. The ATP-dependent interaction of eukaryotic initiation factors with mRNA. J Biol Chem. 1987 Mar 15;262(8):3826–3832. [PubMed] [Google Scholar]
  2. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  3. Burgess S., Couto J. R., Guthrie C. A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing. Cell. 1990 Mar 9;60(5):705–717. doi: 10.1016/0092-8674(90)90086-t. [DOI] [PubMed] [Google Scholar]
  4. Company M., Arenas J., Abelson J. Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature. 1991 Feb 7;349(6309):487–493. doi: 10.1038/349487a0. [DOI] [PubMed] [Google Scholar]
  5. Dalbadie-McFarland G., Abelson J. PRP5: a helicase-like protein required for mRNA splicing in yeast. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4236–4240. doi: 10.1073/pnas.87.11.4236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dreyfuss G., Matunis M. J., Piñol-Roma S., Burd C. G. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. 1993;62:289–321. doi: 10.1146/annurev.bi.62.070193.001445. [DOI] [PubMed] [Google Scholar]
  7. Fernández A., Laín S., García J. A. RNA helicase activity of the plum pox potyvirus CI protein expressed in Escherichia coli. Mapping of an RNA binding domain. Nucleic Acids Res. 1995 Apr 25;23(8):1327–1332. doi: 10.1093/nar/23.8.1327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ford M. J., Anton I. A., Lane D. P. Nuclear protein with sequence homology to translation initiation factor eIF-4A. Nature. 1988 Apr 21;332(6166):736–738. doi: 10.1038/332736a0. [DOI] [PubMed] [Google Scholar]
  9. Fuller-Pace F. V., Nicol S. M., Reid A. D., Lane D. P. DbpA: a DEAD box protein specifically activated by 23s rRNA. EMBO J. 1993 Sep;12(9):3619–3626. doi: 10.1002/j.1460-2075.1993.tb06035.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ghisolfi L., Joseph G., Amalric F., Erard M. The glycine-rich domain of nucleolin has an unusual supersecondary structure responsible for its RNA-helix-destabilizing properties. J Biol Chem. 1992 Feb 15;267(5):2955–2959. [PubMed] [Google Scholar]
  11. Gibson T. J., Thompson J. D. Detection of dsRNA-binding domains in RNA helicase A and Drosophila maleless: implications for monomeric RNA helicases. Nucleic Acids Res. 1994 Jul 11;22(13):2552–2556. doi: 10.1093/nar/22.13.2552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Girard J. P., Bagni C., Caizergues-Ferrer M., Amalric F., Lapeyre B. Identification of a segment of the small nucleolar ribonucleoprotein-associated protein GAR1 that is sufficient for nucleolar accumulation. J Biol Chem. 1994 Jul 15;269(28):18499–18506. [PubMed] [Google Scholar]
  13. Girard J. P., Lehtonen H., Caizergues-Ferrer M., Amalric F., Tollervey D., Lapeyre B. GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast. EMBO J. 1992 Feb;11(2):673–682. doi: 10.1002/j.1460-2075.1992.tb05099.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Gururajan R., Mathews L., Longo F. J., Weeks D. L. An3 mRNA encodes an RNA helicase that colocalizes with nucleoli in Xenopus oocytes in a stage-specific manner. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2056–2060. doi: 10.1073/pnas.91.6.2056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gururajan R., Perry-O'Keefe H., Melton D. A., Weeks D. L. The Xenopus localized messenger RNA An3 may encode an ATP-dependent RNA helicase. Nature. 1991 Feb 21;349(6311):717–719. doi: 10.1038/349717a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hirling H., Scheffner M., Restle T., Stahl H. RNA helicase activity associated with the human p68 protein. Nature. 1989 Jun 15;339(6225):562–564. doi: 10.1038/339562a0. [DOI] [PubMed] [Google Scholar]
  19. Hodgman T. C. A new superfamily of replicative proteins. Nature. 1988 May 5;333(6168):22–23. doi: 10.1038/333022b0. [DOI] [PubMed] [Google Scholar]
  20. Howard E. A., Zupan J. R., Citovsky V., Zambryski P. C. The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells. Cell. 1992 Jan 10;68(1):109–118. doi: 10.1016/0092-8674(92)90210-4. [DOI] [PubMed] [Google Scholar]
  21. Iggo R. D., Jamieson D. J., MacNeill S. A., Southgate J., McPheat J., Lane D. P. p68 RNA helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts. Mol Cell Biol. 1991 Mar;11(3):1326–1333. doi: 10.1128/mcb.11.3.1326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Iost I., Dreyfus M. mRNAs can be stabilized by DEAD-box proteins. Nature. 1994 Nov 10;372(6502):193–196. doi: 10.1038/372193a0. [DOI] [PubMed] [Google Scholar]
  23. Itadani H., Sugita M., Sugiura M. Structure and expression of a cDNA encoding an RNA helicase-like protein in tobacco. Plant Mol Biol. 1994 Jan;24(1):249–252. doi: 10.1007/BF00040593. [DOI] [PubMed] [Google Scholar]
  24. Jong A. Y., Clark M. W., Gilbert M., Oehm A., Campbell J. L. Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins. Mol Cell Biol. 1987 Aug;7(8):2947–2955. doi: 10.1128/mcb.7.8.2947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kalderon D., Richardson W. D., Markham A. F., Smith A. E. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature. 1984 Sep 6;311(5981):33–38. doi: 10.1038/311033a0. [DOI] [PubMed] [Google Scholar]
  26. Kiledjian M., Dreyfuss G. Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box. EMBO J. 1992 Jul;11(7):2655–2664. doi: 10.1002/j.1460-2075.1992.tb05331.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Koop H. U., Steinmüller K., Wagner H., Rössler C., Eibl C., Sacher L. Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta. 1996;199(2):193–201. doi: 10.1007/BF00196559. [DOI] [PubMed] [Google Scholar]
  28. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Lapeyre B., Bourbon H., Amalric F. Nucleolin, the major nucleolar protein of growing eukaryotic cells: an unusual protein structure revealed by the nucleotide sequence. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1472–1476. doi: 10.1073/pnas.84.6.1472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lapeyre B., Mariottini P., Mathieu C., Ferrer P., Amaldi F., Amalric F., Caizergues-Ferrer M. Molecular cloning of Xenopus fibrillarin, a conserved U3 small nuclear ribonucleoprotein recognized by antisera from humans with autoimmune disease. Mol Cell Biol. 1990 Jan;10(1):430–434. doi: 10.1128/mcb.10.1.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lasko P. F., Ashburner M. The product of the Drosophila gene vasa is very similar to eukaryotic initiation factor-4A. Nature. 1988 Oct 13;335(6191):611–617. doi: 10.1038/335611a0. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Laín S., Riechmann J. L., García J. A. RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus. Nucleic Acids Res. 1990 Dec 11;18(23):7003–7006. doi: 10.1093/nar/18.23.7003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lee C. G., Hurwitz J. A new RNA helicase isolated from HeLa cells that catalytically translocates in the 3' to 5' direction. J Biol Chem. 1992 Mar 5;267(7):4398–4407. [PubMed] [Google Scholar]
  36. Lee W. C., Xue Z. X., Mélèse T. The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs. J Cell Biol. 1991 Apr;113(1):1–12. doi: 10.1083/jcb.113.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Liang S., Hitomi M., Hu Y. H., Liu Y., Tartakoff A. M. A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA. Mol Cell Biol. 1996 Sep;16(9):5139–5146. doi: 10.1128/mcb.16.9.5139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Linder P., Lasko P. F., Ashburner M., Leroy P., Nielsen P. J., Nishi K., Schnier J., Slonimski P. P. Birth of the D-E-A-D box. Nature. 1989 Jan 12;337(6203):121–122. doi: 10.1038/337121a0. [DOI] [PubMed] [Google Scholar]
  39. Lorković Z. J., Schröder W. P., Pakrasi H. B., Irrgang K. D., Herrmann R. G., Oelmüller R. Molecular characterization of PsbW, a nuclear-encoded component of the photosystem II reaction center complex in spinach. Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8930–8934. doi: 10.1073/pnas.92.19.8930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Margossian S. P., Li H., Zassenhaus H. P., Butow R. A. The DExH box protein Suv3p is a component of a yeast mitochondrial 3'-to-5' exoribonuclease that suppresses group I intron toxicity. Cell. 1996 Jan 26;84(2):199–209. doi: 10.1016/s0092-8674(00)80975-7. [DOI] [PubMed] [Google Scholar]
  41. Mattaj I. W. RNA recognition: a family matter? Cell. 1993 Jun 4;73(5):837–840. doi: 10.1016/0092-8674(93)90265-r. [DOI] [PubMed] [Google Scholar]
  42. Messmer B., Dreyer C. Requirements for nuclear translocation and nucleolar accumulation of nucleolin of Xenopus laevis. Eur J Cell Biol. 1993 Aug;61(2):369–382. [PubMed] [Google Scholar]
  43. Milburn S. C., Hershey J. W., Davies M. V., Kelleher K., Kaufman R. J. Cloning and expression of eukaryotic initiation factor 4B cDNA: sequence determination identifies a common RNA recognition motif. EMBO J. 1990 Sep;9(9):2783–2790. doi: 10.1002/j.1460-2075.1990.tb07466.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. O'Day C. L., Chavanikamannil F., Abelson J. 18S rRNA processing requires the RNA helicase-like protein Rrp3. Nucleic Acids Res. 1996 Aug 15;24(16):3201–3207. doi: 10.1093/nar/24.16.3201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ono Y., Ohno M., Shimura Y. Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22. Mol Cell Biol. 1994 Nov;14(11):7611–7620. doi: 10.1128/mcb.14.11.7611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Owttrim G. W., Hofmann S., Kuhlemeier C. Divergent genes for translation initiation factor eIF-4A are coordinately expressed in tobacco. Nucleic Acids Res. 1991 Oct 25;19(20):5491–5496. doi: 10.1093/nar/19.20.5491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pause A., Méthot N., Sonenberg N. The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis. Mol Cell Biol. 1993 Nov;13(11):6789–6798. doi: 10.1128/mcb.13.11.6789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Pause A., Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992 Jul;11(7):2643–2654. doi: 10.1002/j.1460-2075.1992.tb05330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Py B., Higgins C. F., Krisch H. M., Carpousis A. J. A DEAD-box RNA helicase in the Escherichia coli RNA degradosome. Nature. 1996 May 9;381(6578):169–172. doi: 10.1038/381169a0. [DOI] [PubMed] [Google Scholar]
  50. Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
  51. Rodríguez P. L., Carrasco L. Poliovirus protein 2C contains two regions involved in RNA binding activity. J Biol Chem. 1995 Apr 28;270(17):10105–10112. doi: 10.1074/jbc.270.17.10105. [DOI] [PubMed] [Google Scholar]
  52. Roussell D. L., Bennett K. L. glh-1, a germ-line putative RNA helicase from Caenorhabditis, has four zinc fingers. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9300–9304. doi: 10.1073/pnas.90.20.9300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Rozen F., Edery I., Meerovitch K., Dever T. E., Merrick W. C., Sonenberg N. Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol. 1990 Mar;10(3):1134–1144. doi: 10.1128/mcb.10.3.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Schimmang T., Tollervey D., Kern H., Frank R., Hurt E. C. A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. EMBO J. 1989 Dec 20;8(13):4015–4024. doi: 10.1002/j.1460-2075.1989.tb08584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Schwer B., Guthrie C. PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature. 1991 Feb 7;349(6309):494–499. doi: 10.1038/349494a0. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Shuman S. Vaccinia virus RNA helicase: an essential enzyme related to the DE-H family of RNA-dependent NTPases. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10935–10939. doi: 10.1073/pnas.89.22.10935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Simpson G. G., Clark G. P., Rothnie H. M., Boelens W., van Venrooij W., Brown J. W. Molecular characterization of the spliceosomal proteins U1A and U2B" from higher plants. EMBO J. 1995 Sep 15;14(18):4540–4550. doi: 10.1002/j.1460-2075.1995.tb00133.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Simpson G. G., Vaux P., Clark G., Waugh R., Beggs J. D., Brown J. W. Evolutionary conservation of the spliceosomal protein, U2B''. Nucleic Acids Res. 1991 Oct 11;19(19):5213–5217. doi: 10.1093/nar/19.19.5213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Snudden D. K., Hearing J., Smith P. R., Grässer F. A., Griffin B. E. EBNA-1, the major nuclear antigen of Epstein-Barr virus, resembles 'RGG' RNA binding proteins. EMBO J. 1994 Oct 17;13(20):4840–4847. doi: 10.1002/j.1460-2075.1994.tb06810.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Strauss E. J., Guthrie C. PRP28, a 'DEAD-box' protein, is required for the first step of mRNA splicing in vitro. Nucleic Acids Res. 1994 Aug 11;22(15):3187–3193. doi: 10.1093/nar/22.15.3187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  64. Séraphin B., Simon M., Boulet A., Faye G. Mitochondrial splicing requires a protein from a novel helicase family. Nature. 1989 Jan 5;337(6202):84–87. doi: 10.1038/337084a0. [DOI] [PubMed] [Google Scholar]
  65. Teigelkamp S., McGarvey M., Plumpton M., Beggs J. D. The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA. EMBO J. 1994 Feb 15;13(4):888–897. doi: 10.1002/j.1460-2075.1994.tb06332.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Valdez B. C., Henning D., Busch R. K., Woods K., Flores-Rozas H., Hurwitz J., Perlaky L., Busch H. A nucleolar RNA helicase recognized by autoimmune antibodies from a patient with watermelon stomach disease. Nucleic Acids Res. 1996 Apr 1;24(7):1220–1224. doi: 10.1093/nar/24.7.1220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wassarman D. A., Steitz J. A. RNA splicing. Alive with DEAD proteins. Nature. 1991 Feb 7;349(6309):463–464. doi: 10.1038/349463a0. [DOI] [PubMed] [Google Scholar]
  68. Xu D., Nouraini S., Field D., Tang S. J., Friesen J. D. An RNA-dependent ATPase associated with U2/U6 snRNAs in pre-mRNA splicing. Nature. 1996 Jun 20;381(6584):709–713. doi: 10.1038/381709a0. [DOI] [PubMed] [Google Scholar]
  69. Zahler A. M., Lane W. S., Stolk J. A., Roth M. B. SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev. 1992 May;6(5):837–847. doi: 10.1101/gad.6.5.837. [DOI] [PubMed] [Google Scholar]

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