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. 1996 Nov 1;24(21):4152–4157. doi: 10.1093/nar/24.21.4152

Inverse splicing of a discontinuous pre-mRNA intron generates a circular exon in a HeLa cell nuclear extract.

S Braun 1, H Domdey 1, K Wiebauer 1
PMCID: PMC146224  PMID: 8932365

Abstract

We have recently reported the first example of inverse splicing of a eukaryotic pre-mRNA intron using a whole cell extract from the yeast Saccharomyces cerevisiae. The concomitant circularization of the exon in the course of this splicing reaction gave rise to the hypothesis that the circular RNA species, which had been recently discovered in vivo in mammalian cells, were generated by inverse splicing. Here we report the formation of a circular exon in HeLa cell nuclear extracts by an inverse splicing reaction of the second intron of the human beta-globin gene from a pre-mRNA transcript in which the two intron halves flanked an artificially fused, single exon. Our data demonstrate that the mammalian pre-mRNA splicing system has indeed an intrinsic capability of aligning splice sites in reverse order and that this alignment can be followed by a complete splicing reaction, whereby the discontinuous intron sequences are removed. Thus we propose that circular exons in vivo arise as a result of an inverse splicing reaction following the pairing of a 5' splice site with an upstream 3' splice site and that the frequency of this event is influenced by the presence and strength of other, competing splice sites.

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Selected References

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  1. Bailleul B. During in vivo maturation of eukaryotic nuclear mRNA, splicing yields excised exon circles. Nucleic Acids Res. 1996 Mar 15;24(6):1015–1019. doi: 10.1093/nar/24.6.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Black D. L. Finding splice sites within a wilderness of RNA. RNA. 1995 Oct;1(8):763–771. [PMC free article] [PubMed] [Google Scholar]
  3. Capel B., Swain A., Nicolis S., Hacker A., Walter M., Koopman P., Goodfellow P., Lovell-Badge R. Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell. 1993 Jun 4;73(5):1019–1030. doi: 10.1016/0092-8674(93)90279-y. [DOI] [PubMed] [Google Scholar]
  4. Chiara M. D., Reed R. A two-step mechanism for 5' and 3' splice-site pairing. Nature. 1995 Jun 8;375(6531):510–513. doi: 10.1038/375510a0. [DOI] [PubMed] [Google Scholar]
  5. Cocquerelle C., Daubersies P., Majérus M. A., Kerckaert J. P., Bailleul B. Splicing with inverted order of exons occurs proximal to large introns. EMBO J. 1992 Mar;11(3):1095–1098. doi: 10.1002/j.1460-2075.1992.tb05148.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cocquerelle C., Mascrez B., Hétuin D., Bailleul B. Mis-splicing yields circular RNA molecules. FASEB J. 1993 Jan;7(1):155–160. doi: 10.1096/fasebj.7.1.7678559. [DOI] [PubMed] [Google Scholar]
  7. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dominski Z., Kole R. Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing. Mol Cell Biol. 1994 Nov;14(11):7445–7454. doi: 10.1128/mcb.14.11.7445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ford E., Ares M., Jr Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3117–3121. doi: 10.1073/pnas.91.8.3117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fu X. D., Mayeda A., Maniatis T., Krainer A. R. General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11224–11228. doi: 10.1073/pnas.89.23.11224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Green M. R. Pre-mRNA splicing. Annu Rev Genet. 1986;20:671–708. doi: 10.1146/annurev.ge.20.120186.003323. [DOI] [PubMed] [Google Scholar]
  12. Hall S. L., Padgett R. A. Conserved sequences in a class of rare eukaryotic nuclear introns with non-consensus splice sites. J Mol Biol. 1994 Jun 10;239(3):357–365. doi: 10.1006/jmbi.1994.1377. [DOI] [PubMed] [Google Scholar]
  13. Hall S. L., Padgett R. A. Requirement of U12 snRNA for in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns. Science. 1996 Mar 22;271(5256):1716–1718. doi: 10.1126/science.271.5256.1716. [DOI] [PubMed] [Google Scholar]
  14. Humphrey M. B., Bryan J., Cooper T. A., Berget S. M. A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon. Mol Cell Biol. 1995 Aug;15(8):3979–3988. doi: 10.1128/mcb.15.8.3979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jarrell K. A. Inverse splicing of a group II intron. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8624–8627. doi: 10.1073/pnas.90.18.8624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jeske Y. W., Bowles J., Greenfield A., Koopman P. Expression of a linear Sry transcript in the mouse genital ridge. Nat Genet. 1995 Aug;10(4):480–482. doi: 10.1038/ng0895-480. [DOI] [PubMed] [Google Scholar]
  17. Konarska M. M., Padgett R. A., Sharp P. A. Trans splicing of mRNA precursors in vitro. Cell. 1985 Aug;42(1):165–171. doi: 10.1016/s0092-8674(85)80112-4. [DOI] [PubMed] [Google Scholar]
  18. Lamond A. I., Sproat B., Ryder U., Hamm J. Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2'-OMe RNA. Cell. 1989 Jul 28;58(2):383–390. doi: 10.1016/0092-8674(89)90852-0. [DOI] [PubMed] [Google Scholar]
  19. Lawn R. M., Efstratiadis A., O'Connell C., Maniatis T. The nucleotide sequence of the human beta-globin gene. Cell. 1980 Oct;21(3):647–651. doi: 10.1016/0092-8674(80)90428-6. [DOI] [PubMed] [Google Scholar]
  20. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nigro J. M., Cho K. R., Fearon E. R., Kern S. E., Ruppert J. M., Oliner J. D., Kinzler K. W., Vogelstein B. Scrambled exons. Cell. 1991 Feb 8;64(3):607–613. doi: 10.1016/0092-8674(91)90244-s. [DOI] [PubMed] [Google Scholar]
  22. Pasman Z., Garcia-Blanco M. A. The 5' and 3' splice sites come together via a three dimensional diffusion mechanism. Nucleic Acids Res. 1996 May 1;24(9):1638–1645. doi: 10.1093/nar/24.9.1638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Puttaraju M., Been M. D. Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons. Nucleic Acids Res. 1992 Oct 25;20(20):5357–5364. doi: 10.1093/nar/20.20.5357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Robberson B. L., Cote G. J., Berget S. M. Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol. 1990 Jan;10(1):84–94. doi: 10.1128/mcb.10.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Schindewolf C. A., Domdey H. Splicing of a circular yeast pre-mRNA in vitro. Nucleic Acids Res. 1995 Apr 11;23(7):1133–1139. doi: 10.1093/nar/23.7.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schindewolf C., Braun S., Domdey H. In vitro generation of a circular exon from a linear pre-mRNA transcript. Nucleic Acids Res. 1996 Apr 1;24(7):1260–1266. doi: 10.1093/nar/24.7.1260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Talerico M., Berget S. M. Intron definition in splicing of small Drosophila introns. Mol Cell Biol. 1994 May;14(5):3434–3445. doi: 10.1128/mcb.14.5.3434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tanaka K., Watakabe A., Shimura Y. Polypurine sequences within a downstream exon function as a splicing enhancer. Mol Cell Biol. 1994 Feb;14(2):1347–1354. doi: 10.1128/mcb.14.2.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tarn W. Y., Steitz J. A. A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro. Cell. 1996 Mar 8;84(5):801–811. doi: 10.1016/s0092-8674(00)81057-0. [DOI] [PubMed] [Google Scholar]
  31. Tarn W. Y., Steitz J. A. SR proteins can compensate for the loss of U1 snRNP functions in vitro. Genes Dev. 1994 Nov 15;8(22):2704–2717. doi: 10.1101/gad.8.22.2704. [DOI] [PubMed] [Google Scholar]
  32. Tian M., Maniatis T. A splicing enhancer exhibits both constitutive and regulated activities. Genes Dev. 1994 Jul 15;8(14):1703–1712. doi: 10.1101/gad.8.14.1703. [DOI] [PubMed] [Google Scholar]
  33. 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]

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