Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 May 15;16(10):2955–2967. doi: 10.1093/emboj/16.10.2955

The human RNA 3'-terminal phosphate cyclase is a member of a new family of proteins conserved in Eucarya, Bacteria and Archaea.

P Genschik 1, E Billy 1, M Swianiewicz 1, W Filipowicz 1
PMCID: PMC1169903  PMID: 9184239

Abstract

RNA 3'-terminal phosphate cyclase catalyses the ATP-dependent conversion of the 3'-phosphate to a 2',3'-cyclic phosphodiester at the end of RNA. The physiological function of the cyclase is not known, but the enzyme could be involved in the maintenance of cyclic ends in tRNA splicing intermediates or in the cyclization of the 3' end of U6 snRNA. In this work, we describe cloning of the human cyclase cDNA. The purified bacterially overexpressed protein underwent adenylylation in the presence of [alpha-32P]ATP and catalysed cyclization of the 3'-terminal phosphate in different RNA substrates, consistent with previous findings. Comparison of oligoribonucleotides and oligodeoxyribonucleotides of identical sequence demonstrated that the latter are approximately 500-fold poorer substrates for the enzyme. In Northern analysis, the cyclase was expressed in all analysed mammalian tissues and cell lines. Indirect immunofluorescence, performed with different transfected mammalian cell lines, showed that this protein is nuclear, with a diffuse nucleoplasmic localization. The sequence of the human cyclase has no apparent motifs in common with any proteins of known function. However, inspection of the databases identified proteins showing strong similarity to the enzyme, originating from as evolutionarily distant organisms as yeast, plants, the bacterium Escherichia coli and the archaeon Methanococcus jannaschii. The overexpressed E. coli protein has cyclase activity similar to that of the human enzyme. The conservation of the RNA 3'-terminal phosphate cyclase among Eucarya, Bacteria and Archaea argues that the enzyme performs an important function in RNA metabolism.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Attardi D. G., Margarit I., Tocchini-Valentini G. P. Structural alterations in mutant precursors of the yeast tRNALeu3 gene which behave as defective substrates for a highly purified splicing endoribonuclease. EMBO J. 1985 Dec 1;4(12):3289–3297. doi: 10.1002/j.1460-2075.1985.tb04079.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Branch A. D., Robertson H. D., Greer C., Gegenheimer P., Peebles C., Abelson J. Cell-free circularization of viroid progeny RNA by an RNA ligase from wheat germ. Science. 1982 Sep 17;217(4565):1147–1149. doi: 10.1126/science.217.4565.1147. [DOI] [PubMed] [Google Scholar]
  3. Bult C. J., White O., Olsen G. J., Zhou L., Fleischmann R. D., Sutton G. G., Blake J. A., FitzGerald L. M., Clayton R. A., Gocayne J. D. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science. 1996 Aug 23;273(5278):1058–1073. doi: 10.1126/science.273.5278.1058. [DOI] [PubMed] [Google Scholar]
  4. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Christiansen K., Knudsen B. R., Westergaard O. The covalent eukaryotic topoisomerase I-DNA intermediate catalyzes pH-dependent hydrolysis and alcoholysis. J Biol Chem. 1994 Apr 15;269(15):11367–11373. [PubMed] [Google Scholar]
  6. Cravchik A., Matus A. A novel strategy for the immunological tagging of cDNA constructs. Gene. 1993 Dec 27;137(1):139–143. doi: 10.1016/0378-1119(93)90262-2. [DOI] [PubMed] [Google Scholar]
  7. Cross S. H., Charlton J. A., Nan X., Bird A. P. Purification of CpG islands using a methylated DNA binding column. Nat Genet. 1994 Mar;6(3):236–244. doi: 10.1038/ng0394-236. [DOI] [PubMed] [Google Scholar]
  8. Efimov V. A., Buryakova A. A., Reverdatto S. V., Chakhmakhcheva O. G., Ovchinnikov YuA Rapid synthesis of long-chain deoxyribooligonucleotides by the N-methylimidazolide phosphotriester method. Nucleic Acids Res. 1983 Dec 10;11(23):8369–8387. doi: 10.1093/nar/11.23.8369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  11. Filipowicz W., Konarska M., Gross H. J., Shatkin A. J. RNA 3'-terminal phosphate cyclase activity and RNA ligation in HeLa cell extract. Nucleic Acids Res. 1983 Mar 11;11(5):1405–1418. doi: 10.1093/nar/11.5.1405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Filipowicz W., Shatkin A. J. Origin of splice junction phosphate in tRNAs processed by HeLa cell extract. Cell. 1983 Feb;32(2):547–557. doi: 10.1016/0092-8674(83)90474-9. [DOI] [PubMed] [Google Scholar]
  13. Filipowicz W., Strugala K., Konarska M., Shatkin A. J. Cyclization of RNA 3'-terminal phosphate by cyclase from HeLa cells proceeds via formation of N(3')pp(5')A activated intermediate. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1316–1320. doi: 10.1073/pnas.82.5.1316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Filipowicz W., Vicente O. RNA 3'-terminal phosphate cyclase from HeLa cells. Methods Enzymol. 1990;181:499–510. doi: 10.1016/0076-6879(90)81147-m. [DOI] [PubMed] [Google Scholar]
  15. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  16. Fraser C. M., Gocayne J. D., White O., Adams M. D., Clayton R. A., Fleischmann R. D., Bult C. J., Kerlavage A. R., Sutton G., Kelley J. M. The minimal gene complement of Mycoplasma genitalium. Science. 1995 Oct 20;270(5235):397–403. doi: 10.1126/science.270.5235.397. [DOI] [PubMed] [Google Scholar]
  17. Furneaux H., Pick L., Hurwitz J. Isolation and characterization of RNA ligase from wheat germ. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3933–3937. doi: 10.1073/pnas.80.13.3933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gegenheimer P., Gabius H. J., Peebles C. L., Abelson J. An RNA ligase from wheat germ which participates in transfer RNA splicing in vitro. J Biol Chem. 1983 Jul 10;258(13):8365–8373. [PubMed] [Google Scholar]
  19. Greer C. L., Javor B., Abelson J. RNA ligase in bacteria: formation of a 2',5' linkage by an E. coli extract. Cell. 1983 Jul;33(3):899–906. doi: 10.1016/0092-8674(83)90032-6. [DOI] [PubMed] [Google Scholar]
  20. Greer C. L., Peebles C. L., Gegenheimer P., Abelson J. Mechanism of action of a yeast RNA ligase in tRNA splicing. Cell. 1983 Feb;32(2):537–546. doi: 10.1016/0092-8674(83)90473-7. [DOI] [PubMed] [Google Scholar]
  21. Hinton D. M., Brennan C. A., Gumport R. I. The preparative synthesis of oligodeoxyribonucleotides using RNA ligase. Nucleic Acids Res. 1982 Mar 25;10(6):1877–1894. doi: 10.1093/nar/10.6.1877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kiberstis P. A., Haseloff J., Zimmern D. 2' phosphomonoester, 3'-5' phosphodiester bond at a unique site in a circular viral RNA. EMBO J. 1985 Mar;4(3):817–822. doi: 10.1002/j.1460-2075.1985.tb03703.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kikuchi Y., Tyc K., Filipowicz W., Sänger H. L., Gross H. J. Circularization of linear viroid RNA via 2'-phosphomonoester, 3', 5'-phosphodiester bonds by a novel type of RNA ligase from wheat germ and Chlamydomonas. Nucleic Acids Res. 1982 Dec 11;10(23):7521–7529. doi: 10.1093/nar/10.23.7521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kimball A. S., Lee J., Jayaram M., Tullius T. D. Sequence-specific cleavage of DNA via nucleophilic attack of hydrogen peroxide, assisted by Flp recombinase. Biochemistry. 1993 May 11;32(18):4698–4701. doi: 10.1021/bi00069a002. [DOI] [PubMed] [Google Scholar]
  25. Kjems J., Garrett R. A. Novel splicing mechanism for the ribosomal RNA intron in the archaebacterium Desulfurococcus mobilis. Cell. 1988 Aug 26;54(5):693–703. doi: 10.1016/s0092-8674(88)80014-x. [DOI] [PubMed] [Google Scholar]
  26. Konarska M., Filipowicz W., Domdey H., Gross H. J. Formation of a 2'-phosphomonoester, 3',5'-phosphodiester linkage by a novel RNA ligase in wheat germ. Nature. 1981 Sep 10;293(5828):112–116. doi: 10.1038/293112a0. [DOI] [PubMed] [Google Scholar]
  27. Konarska M., Filipowicz W., Gross H. J. RNA ligation via 2'-phosphomonoester, 3'5'-phosphodiester linkage: requirement of 2',3'-cyclic phosphate termini and involvement of a 5'-hydroxyl polynucleotide kinase. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1474–1478. doi: 10.1073/pnas.79.5.1474. [DOI] [PMC free article] [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. Laski F. A., Fire A. Z., RajBhandary U. L., Sharp P. A. Characterization of tRNA precursor splicing in mammalian extracts. J Biol Chem. 1983 Oct 10;258(19):11974–11980. [PubMed] [Google Scholar]
  30. Latham K. A., Lloyd R. S. Delta-elimination by T4 endonuclease V at a thymine dimer site requires a secondary binding event and amino acid Glu-23. Biochemistry. 1995 Jul 11;34(27):8796–8803. doi: 10.1021/bi00027a031. [DOI] [PubMed] [Google Scholar]
  31. Lund E., Dahlberg J. E. Cyclic 2',3'-phosphates and nontemplated nucleotides at the 3' end of spliceosomal U6 small nuclear RNA's. Science. 1992 Jan 17;255(5042):327–330. doi: 10.1126/science.1549778. [DOI] [PubMed] [Google Scholar]
  32. Manley J. L., Tacke R. SR proteins and splicing control. Genes Dev. 1996 Jul 1;10(13):1569–1579. doi: 10.1101/gad.10.13.1569. [DOI] [PubMed] [Google Scholar]
  33. Peebles C. L., Gegenheimer P., Abelson J. Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell. 1983 Feb;32(2):525–536. doi: 10.1016/0092-8674(83)90472-5. [DOI] [PubMed] [Google Scholar]
  34. Perkins K. K., Furneaux H., Hurwitz J. Isolation and characterization of an RNA ligase from HeLa cells. Proc Natl Acad Sci U S A. 1985 Feb;82(3):684–688. doi: 10.1073/pnas.82.3.684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Phizicky E. M., Consaul S. A., Nehrke K. W., Abelson J. Yeast tRNA ligase mutants are nonviable and accumulate tRNA splicing intermediates. J Biol Chem. 1992 Mar 5;267(7):4577–4582. [PubMed] [Google Scholar]
  36. Phizicky E. M., Greer C. L. Pre-tRNA splicing: variation on a theme or exception to the rule? Trends Biochem Sci. 1993 Jan;18(1):31–34. doi: 10.1016/0968-0004(93)90085-2. [DOI] [PubMed] [Google Scholar]
  37. Phizicky E. M., Schwartz R. C., Abelson J. Saccharomyces cerevisiae tRNA ligase. Purification of the protein and isolation of the structural gene. J Biol Chem. 1986 Feb 25;261(6):2978–2986. [PubMed] [Google Scholar]
  38. Pick L., Furneaux H., Hurwitz J. Purification of wheat germ RNA ligase. II. Mechanism of action of wheat germ RNA ligase. J Biol Chem. 1986 May 25;261(15):6694–6704. [PubMed] [Google Scholar]
  39. Rand K. N., Gait M. J. Sequence and cloning of bacteriophage T4 gene 63 encoding RNA ligase and tail fibre attachment activities. EMBO J. 1984 Feb;3(2):397–402. doi: 10.1002/j.1460-2075.1984.tb01819.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rauhut R., Green P. R., Abelson J. Yeast tRNA-splicing endonuclease is a heterotrimeric enzyme. J Biol Chem. 1990 Oct 25;265(30):18180–18184. [PubMed] [Google Scholar]
  41. Reinberg D., Arenas J., Hurwitz J. The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives. J Biol Chem. 1985 May 25;260(10):6088–6097. [PubMed] [Google Scholar]
  42. Richards G. M., Laskowski M., Sr Negative charge at the 3' terminus of oligonucleotides and resistance to venom exonuclease. Biochemistry. 1969 May;8(5):1786–1795. doi: 10.1021/bi00833a002. [DOI] [PubMed] [Google Scholar]
  43. Sadowski P. D. The Flp recombinase of the 2-microns plasmid of Saccharomyces cerevisiae. Prog Nucleic Acid Res Mol Biol. 1995;51:53–91. [PubMed] [Google Scholar]
  44. Schwartz R. C., Greer C. L., Gegenheimer P., Abelson J. Enzymatic mechanism of an RNA ligase from wheat germ. J Biol Chem. 1983 Jul 10;258(13):8374–8383. [PubMed] [Google Scholar]
  45. Shuman S., Schwer B. RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases. Mol Microbiol. 1995 Aug;17(3):405–410. doi: 10.1111/j.1365-2958.1995.mmi_17030405.x. [DOI] [PubMed] [Google Scholar]
  46. Stange N., Beier H. A cell-free plant extract for accurate pre-tRNA processing, splicing and modification. EMBO J. 1987 Sep;6(9):2811–2818. doi: 10.1002/j.1460-2075.1987.tb02577.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tazi J., Forne T., Jeanteur P., Cathala G., Brunel C. Mammalian U6 small nuclear RNA undergoes 3' end modifications within the spliceosome. Mol Cell Biol. 1993 Mar;13(3):1641–1650. doi: 10.1128/mcb.13.3.1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Thompson L. D., Daniels C. J. A tRNA(Trp) intron endonuclease from Halobacterium volcanii. Unique substrate recognition properties. J Biol Chem. 1988 Dec 5;263(34):17951–17959. [PubMed] [Google Scholar]
  50. Vicente O., Filipowicz W. Purification of RNA 3'-terminal phosphate cyclase from HeLa cells. Covalent modification of the enzyme with different nucleotides. Eur J Biochem. 1988 Sep 15;176(2):431–439. doi: 10.1111/j.1432-1033.1988.tb14300.x. [DOI] [PubMed] [Google Scholar]
  51. Zillmann M., Gorovsky M. A., Phizicky E. M. Conserved mechanism of tRNA splicing in eukaryotes. Mol Cell Biol. 1991 Nov;11(11):5410–5416. doi: 10.1128/mcb.11.11.5410. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES