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. 2000 Apr;6(4):616–627. doi: 10.1017/s1355838200992203

Expression of the Naegleria intron endonuclease is dependent on a functional group I self-cleaving ribozyme.

W A Decatur 1, S Johansen 1, V M Vogt 1
PMCID: PMC1369942  PMID: 10786852

Abstract

NaSSU1 is a complex nuclear group I intron found in several species of Naegleria, consisting of a large self-splicing group I ribozyme (NaGIR2), which itself is interrupted by a small, group I-like ribozyme (NaGIR1) and an open reading frame (ORF) coding for a homing endonuclease. The GIR1 ribozyme cleaves in vitro transcripts of NaSSU1 at two internal processing sites about 400 nt downstream of the 5' end of the intron, proximal to the endonuclease ORF. Here we demonstrate that self-cleavage of the excised intron also occurs in vivo in Naegleria gruberi, generating an ORF-containing RNA that possesses a short leader with a sequence element likely to be involved in gene expression. To assess the functional significance of self-cleavage, we constructed a genetic system in Saccharomyces cerevisiae. First, a mutant yeast strain was selected with a mutation in all the rRNA genes, rendering the rDNA resistant to cleavage by the Naegleria endonuclease. Active endonuclease, which is otherwise lethal, could be expressed readily in these cells. Endonuclease activity also could be detected in extracts of yeast harboring plasmids in which the endonuclease ORF was embedded in its native context in the intron. Analysis of the RNA from these yeast cells showed that the excised intron RNA was processed as in N. gruberi. A mutant intron constructed to prevent self-cleavage of the RNA failed to express endonuclease activity. These results support the hypothesis that the NaGIR1-catalyzed self-cleavage of the intron RNA is a key event in expression of the endonuclease.

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Selected References

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

  1. Belfort M., Roberts R. J. Homing endonucleases: keeping the house in order. Nucleic Acids Res. 1997 Sep 1;25(17):3379–3388. doi: 10.1093/nar/25.17.3379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Belsham G. J., Sonenberg N. RNA-protein interactions in regulation of picornavirus RNA translation. Microbiol Rev. 1996 Sep;60(3):499–511. doi: 10.1128/mr.60.3.499-511.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cech T. R., Damberger S. H., Gutell R. R. Representation of the secondary and tertiary structure of group I introns. Nat Struct Biol. 1994 May;1(5):273–280. doi: 10.1038/nsb0594-273. [DOI] [PubMed] [Google Scholar]
  4. Cech T. R. Self-splicing of group I introns. Annu Rev Biochem. 1990;59:543–568. doi: 10.1146/annurev.bi.59.070190.002551. [DOI] [PubMed] [Google Scholar]
  5. Cherest H., Thomas D., Surdin-Kerjan Y. Nucleotide sequence of the MET8 gene of Saccharomyces cerevisiae. Nucleic Acids Res. 1990 Feb 11;18(3):659–659. doi: 10.1093/nar/18.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dalgaard J. Z., Garrett R. A., Belfort M. A site-specific endonuclease encoded by a typical archaeal intron. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5414–5417. doi: 10.1073/pnas.90.12.5414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. De Jonckheere J. F., Brown S. Loss of the ORF in the SSUrDNA group I intron of one Naegleria lineage. Nucleic Acids Res. 1994 Sep 25;22(19):3925–3927. doi: 10.1093/nar/22.19.3925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. De Jonckheere J. F. Evidence for the ancestral origin of group I introns in the SSUrDNA of Naegleria spp. J Eukaryot Microbiol. 1994 Sep-Oct;41(5):457–463. doi: 10.1111/j.1550-7408.1994.tb06042.x. [DOI] [PubMed] [Google Scholar]
  9. Decatur W. A., Einvik C., Johansen S., Vogt V. M. Two group I ribozymes with different functions in a nuclear rDNA intron. EMBO J. 1995 Sep 15;14(18):4558–4568. doi: 10.1002/j.1460-2075.1995.tb00135.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dürrenberger F., Rochaix J. D. Chloroplast ribosomal intron of Chlamydomonas reinhardtii: in vitro self-splicing, DNA endonuclease activity and in vivo mobility. EMBO J. 1991 Nov;10(11):3495–3501. doi: 10.1002/j.1460-2075.1991.tb04913.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Einvik C., Decatur W. A., Embley T. M., Vogt V. M., Johansen S. Naegleria nucleolar introns contain two group I ribozymes with different functions in RNA splicing and processing. RNA. 1997 Jul;3(7):710–720. [PMC free article] [PubMed] [Google Scholar]
  12. Einvik C., Elde M., Johansen S. Group I twintrons: genetic elements in myxomycete and schizopyrenid amoeboflagellate ribosomal DNAs. J Biotechnol. 1998 Sep 17;64(1):63–74. doi: 10.1016/s0168-1656(98)00104-7. [DOI] [PubMed] [Google Scholar]
  13. Einvik C., Nielsen H., Westhof E., Michel F., Johansen S. Group I-like ribozymes with a novel core organization perform obligate sequential hydrolytic cleavages at two processing sites. RNA. 1998 May;4(5):530–541. doi: 10.1017/s1355838298971758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Elde M., Haugen P., Willassen N. P., Johansen S. I-NjaI, a nuclear intron-encoded homing endonuclease from Naegleria, generates a pentanucleotide 3' cleavage-overhang within a 19 base-pair partially symmetric DNA recognition site. Eur J Biochem. 1999 Jan;259(1-2):281–288. doi: 10.1046/j.1432-1327.1999.00035.x. [DOI] [PubMed] [Google Scholar]
  15. Good L., Elela S. A., Nazar R. N. Tetrahymena ribozyme disrupts rRNA processing in yeast. J Biol Chem. 1994 Sep 2;269(35):22169–22172. [PubMed] [Google Scholar]
  16. Jabri E., Aigner S., Cech T. R. Kinetic and secondary structure analysis of Naegleria andersoni GIR1, a group I ribozyme whose putative biological function is site-specific hydrolysis. Biochemistry. 1997 Dec 23;36(51):16345–16354. doi: 10.1021/bi9718595. [DOI] [PubMed] [Google Scholar]
  17. Jabri E., Cech T. R. In vitro selection of the Naegleria GIR1 ribozyme identifies three base changes that dramatically improve activity. RNA. 1998 Dec;4(12):1481–1492. doi: 10.1017/s1355838298981237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johansen S., Elde M., Vader A., Haugen P., Haugli K., Haugli F. In vivo mobility of a group I twintron in nuclear ribosomal DNA of the myxomycete Didymium iridis. Mol Microbiol. 1997 May;24(4):737–745. doi: 10.1046/j.1365-2958.1997.3921743.x. [DOI] [PubMed] [Google Scholar]
  19. Johansen S., Vogt V. M. An intron in the nuclear ribosomal DNA of Didymium iridis codes for a group I ribozyme and a novel ribozyme that cooperate in self-splicing. Cell. 1994 Feb 25;76(4):725–734. doi: 10.1016/0092-8674(94)90511-8. [DOI] [PubMed] [Google Scholar]
  20. Johnson A. W. Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively. Mol Cell Biol. 1997 Oct;17(10):6122–6130. doi: 10.1128/mcb.17.10.6122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lambowitz A. M., Perlman P. S. Involvement of aminoacyl-tRNA synthetases and other proteins in group I and group II intron splicing. Trends Biochem Sci. 1990 Nov;15(11):440–444. doi: 10.1016/0968-0004(90)90283-h. [DOI] [PubMed] [Google Scholar]
  22. Lin J., Vogt V. M. I-PpoI, the endonuclease encoded by the group I intron PpLSU3, is expressed from an RNA polymerase I transcript. Mol Cell Biol. 1998 Oct;18(10):5809–5817. doi: 10.1128/mcb.18.10.5809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Muscarella D. E., Ellison E. L., Ruoff B. M., Vogt V. M. Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum. Mol Cell Biol. 1990 Jul;10(7):3386–3396. doi: 10.1128/mcb.10.7.3386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Muscarella D. E., Vogt V. M. A mobile group I intron from Physarum polycephalum can insert itself and induce point mutations in the nuclear ribosomal DNA of saccharomyces cerevisiae. Mol Cell Biol. 1993 Feb;13(2):1023–1033. doi: 10.1128/mcb.13.2.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Muscarella D. E., Vogt V. M. A mobile group I intron in the nuclear rDNA of Physarum polycephalum. Cell. 1989 Feb 10;56(3):443–454. doi: 10.1016/0092-8674(89)90247-x. [DOI] [PubMed] [Google Scholar]
  26. Nogi Y., Vu L., Nomura M. An approach for isolation of mutants defective in 35S ribosomal RNA synthesis in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7026–7030. doi: 10.1073/pnas.88.16.7026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nogi Y., Yano R., Nomura M. Synthesis of large rRNAs by RNA polymerase II in mutants of Saccharomyces cerevisiae defective in RNA polymerase I. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3962–3966. doi: 10.1073/pnas.88.9.3962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Preiss T., Hentze M. W. Dual function of the messenger RNA cap structure in poly(A)-tail-promoted translation in yeast. Nature. 1998 Apr 2;392(6675):516–520. doi: 10.1038/33192. [DOI] [PubMed] [Google Scholar]
  29. Sachs A. B., Sarnow P., Hentze M. W. Starting at the beginning, middle, and end: translation initiation in eukaryotes. Cell. 1997 Jun 13;89(6):831–838. doi: 10.1016/s0092-8674(00)80268-8. [DOI] [PubMed] [Google Scholar]
  30. Shaw L. C., Lewin A. S. The Cbp2 protein stimulates the splicing of the omega intron of yeast mitochondria. Nucleic Acids Res. 1997 Apr 15;25(8):1597–1604. doi: 10.1093/nar/25.8.1597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vader A., Nielsen H., Johansen S. In vivo expression of the nucleolar group I intron-encoded I-dirI homing endonuclease involves the removal of a spliceosomal intron. EMBO J. 1999 Feb 15;18(4):1003–1013. doi: 10.1093/emboj/18.4.1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Walsh C., Fulton C. Transcription during cell differentiation in Naegleria gruberi. Preferential synthesis of messenger RNA. Biochim Biophys Acta. 1973 Jun 8;312(1):52–71. doi: 10.1016/0005-2787(73)90052-x. [DOI] [PubMed] [Google Scholar]
  34. Wenzlau J. M., Saldanha R. J., Butow R. A., Perlman P. S. A latent intron-encoded maturase is also an endonuclease needed for intron mobility. Cell. 1989 Feb 10;56(3):421–430. doi: 10.1016/0092-8674(89)90245-6. [DOI] [PubMed] [Google Scholar]

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