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. 1996 Apr 1;24(7):1260–1266. doi: 10.1093/nar/24.7.1260

In vitro generation of a circular exon from a linear pre-mRNA transcript.

C Schindewolf 1, S Braun 1, H Domdey 1
PMCID: PMC145787  PMID: 8614628

Abstract

Recent findings have firmly established the existence of circular exons in vivo. We were interested in the possible splicing mechanism by which these unusual mRNA molecules could be created in vitro, though no biological relevance has been attached to their existence as yet. In this report we demonstrate that a modified synthetic linear yeast ACT1 transcript whose sequence begins with the 3'-part of its original intron, is continued by 247 nt of exon sequence and terminates with the 5'-part of its intron will generate a circular exon when introduced to standard in vitro splicing reactions in whole cell splice extracts from Saccharomyces cerevisiae. The formation of a circular exon was found to be independent of specific circular or secondary structures of the pre-mRNA transcript. We hypothesize that circular exons which are found in vivo may be generated from pre-mRNAs which derive from rare events of transcription initiation within an intron.

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Selected References

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  1. Ben-David Y., Bani M. R., Chabot B., De Koven A., Bernstein A. Retroviral insertions downstream of the heterogeneous nuclear ribonucleoprotein A1 gene in erythroleukemia cells: evidence that A1 is not essential for cell growth. Mol Cell Biol. 1992 Oct;12(10):4449–4455. doi: 10.1128/mcb.12.10.4449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birkeland M. L., Johnson P., Trowbridge I. S., Puré E. Changes in CD45 isoform expression accompany antigen-induced murine T-cell activation. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6734–6738. doi: 10.1073/pnas.86.17.6734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blencowe B. J., Nickerson J. A., Issner R., Penman S., Sharp P. A. Association of nuclear matrix antigens with exon-containing splicing complexes. J Cell Biol. 1994 Nov;127(3):593–607. doi: 10.1083/jcb.127.3.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Chen C. Y., Sarnow P. Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science. 1995 Apr 21;268(5209):415–417. doi: 10.1126/science.7536344. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Cáceres J. F., Stamm S., Helfman D. M., Krainer A. R. Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. Science. 1994 Sep 16;265(5179):1706–1709. doi: 10.1126/science.8085156. [DOI] [PubMed] [Google Scholar]
  9. Domdey H., Apostol B., Lin R. J., Newman A., Brody E., Abelson J. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell. 1984 Dec;39(3 Pt 2):611–621. doi: 10.1016/0092-8674(84)90468-9. [DOI] [PubMed] [Google Scholar]
  10. Duchêne M., Löw A., Schweizer A., Domdey H. Molecular consequences of truncations of the first exon for in vitro splicing of yeast actin pre-mRNA. Nucleic Acids Res. 1988 Aug 11;16(15):7233–7239. doi: 10.1093/nar/16.15.7233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Frank D., Guthrie C. An essential splicing factor, SLU7, mediates 3' splice site choice in yeast. Genes Dev. 1992 Nov;6(11):2112–2124. doi: 10.1101/gad.6.11.2112. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Jarrell K. A., Dietrich R. C., Perlman P. S. Group II intron domain 5 facilitates a trans-splicing reaction. Mol Cell Biol. 1988 Jun;8(6):2361–2366. doi: 10.1128/mcb.8.6.2361. [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. 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]
  17. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Köhrer K., Domdey H. Splicing and spliceosome formation of the yeast MATa1 transcript require a minimum distance from the 5' splice site to the internal branch acceptor site. Nucleic Acids Res. 1988 Oct 25;16(20):9457–9475. doi: 10.1093/nar/16.20.9457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Köhrer K., Domdey H. Splicing and spliceosome formation of the yeast MATa1 transcript require a minimum distance from the 5' splice site to the internal branch acceptor site. Nucleic Acids Res. 1988 Oct 25;16(20):9457–9475. doi: 10.1093/nar/16.20.9457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leff S. E., Evans R. M., Rosenfeld M. G. Splice commitment dictates neuron-specific alternative RNA processing in calcitonin/CGRP gene expression. Cell. 1987 Feb 13;48(3):517–524. doi: 10.1016/0092-8674(87)90202-9. [DOI] [PubMed] [Google Scholar]
  21. Lin R. J., Newman A. J., Cheng S. C., Abelson J. Yeast mRNA splicing in vitro. J Biol Chem. 1985 Nov 25;260(27):14780–14792. [PubMed] [Google Scholar]
  22. Lossky M., Anderson G. J., Jackson S. P., Beggs J. Identification of a yeast snRNP protein and detection of snRNP-snRNP interactions. Cell. 1987 Dec 24;51(6):1019–1026. doi: 10.1016/0092-8674(87)90588-5. [DOI] [PubMed] [Google Scholar]
  23. Mayeda A., Helfman D. M., Krainer A. R. Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF. Mol Cell Biol. 1993 May;13(5):2993–3001. doi: 10.1128/mcb.13.5.2993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Nadal-Ginard B., Smith C. W., Patton J. G., Breitbart R. E. Alternative splicing is an efficient mechanism for the generation of protein diversity: contractile protein genes as a model system. Adv Enzyme Regul. 1991;31:261–286. doi: 10.1016/0065-2571(91)90017-g. [DOI] [PubMed] [Google Scholar]
  26. Ng R., Abelson J. Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3912–3916. doi: 10.1073/pnas.77.7.3912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Puttaraju M., Perrotta A. T., Been M. D. A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. 1993 Sep 11;21(18):4253–4258. doi: 10.1093/nar/21.18.4253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ralph D., Huang J., Van der Ploeg L. H. Physical identification of branched intron side-products of splicing in Trypanosoma brucei. EMBO J. 1988 Aug;7(8):2539–2545. doi: 10.1002/j.1460-2075.1988.tb03102.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ruby S. W., Abelson J. Pre-mRNA splicing in yeast. Trends Genet. 1991 Mar;7(3):79–85. doi: 10.1016/0168-9525(91)90276-V. [DOI] [PubMed] [Google Scholar]
  31. Ruskin B., Green M. R. An RNA processing activity that debranches RNA lariats. Science. 1985 Jul 12;229(4709):135–140. doi: 10.1126/science.2990042. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Solnick D. Trans splicing of mRNA precursors. Cell. 1985 Aug;42(1):157–164. doi: 10.1016/s0092-8674(85)80111-2. [DOI] [PubMed] [Google Scholar]
  34. Strubin M., Berte C., Mach B. Alternative splicing and alternative initiation of translation explain the four forms of the Ia antigen-associated invariant chain. EMBO J. 1986 Dec 20;5(13):3483–3488. doi: 10.1002/j.1460-2075.1986.tb04673.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tabaczewski P., Shirwan H., Lewis K., Stroynowski I. Alternative splicing of class Ib major histocompatibility complex transcripts in vivo leads to the expression of soluble Qa-2 molecules in murine blood. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1883–1887. doi: 10.1073/pnas.91.5.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Thompson-Jäger S., Domdey H. Yeast pre-mRNA splicing requires a minimum distance between the 5' splice site and the internal branch acceptor site. Mol Cell Biol. 1987 Nov;7(11):4010–4016. doi: 10.1128/mcb.7.11.4010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Thompson-Jäger S., Domdey H. Yeast pre-mRNA splicing requires a minimum distance between the 5' splice site and the internal branch acceptor site. Mol Cell Biol. 1987 Nov;7(11):4010–4016. doi: 10.1128/mcb.7.11.4010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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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