Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1987 Nov 25;15(22):9279–9298. doi: 10.1093/nar/15.22.9279

The secondary structure of a messenger RNA precursor probed with psoralen is melted in an in vitro splicing reaction.

P L Wollenzien 1, P Goswami 1, J Teare 1, J Szeberenyi 1, C J Goldenberg 1
PMCID: PMC306468  PMID: 3317279

Abstract

The secondary structure of the SP6/mouse insulin precursor RNA was determined by psoralen cross-linking experiments. A series of long-range contacts occur within the left half of the pre-mRNA that contains the intervening sequence. Multiple secondary structures for the pre-mRNA exist since some of the interactions share common sites. In splicing buffer but without the splicing extract added, many of these interactions are stable up to at least 50 degrees C. These interactions, however, are dissociated during the in vitro splicing reaction. This dissociation requires ATP and it occurs during the first 30 min. of the splicing reaction. Pre-mRNAs containing psoralen cross-links in different locations within the RNA molecule were purified and used as substrates for in vitro splicing. Psoralen cross-links at any of the double-stranded regions resulted in complete inhibition of the splicing reaction. This indicates that destabilization of the secondary structure of the SP6/mouse insulin pre-mRNA is necessary for in vitro splicing.

Full text

PDF
9279

Images in this article

9282Image
on p.9282
9283Image
on p.9283
9285Image
on p.9285
9288Image
on p.9288
9290Image
on p.9290
9291Image
on p.9291

Selected References

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

  1. Bass B. L., Weintraub H. A developmentally regulated activity that unwinds RNA duplexes. Cell. 1987 Feb 27;48(4):607–613. doi: 10.1016/0092-8674(87)90239-x. [DOI] [PubMed] [Google Scholar]
  2. Calvet J. P., Pederson T. Base-pairing interactions between small nuclear RNAs and nuclear RNA precursors as revealed by psoralen cross-linking in vivo. Cell. 1981 Nov;26(3 Pt 1):363–370. doi: 10.1016/0092-8674(81)90205-1. [DOI] [PubMed] [Google Scholar]
  3. Choi Y. D., Grabowski P. J., Sharp P. A., Dreyfuss G. Heterogeneous nuclear ribonucleoproteins: role in RNA splicing. Science. 1986 Mar 28;231(4745):1534–1539. doi: 10.1126/science.3952495. [DOI] [PubMed] [Google Scholar]
  4. Cimino G. D., Gamper H. B., Isaacs S. T., Hearst J. E. Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry, and biochemistry. Annu Rev Biochem. 1985;54:1151–1193. doi: 10.1146/annurev.bi.54.070185.005443. [DOI] [PubMed] [Google Scholar]
  5. DiMaria P. R., Kaltwasser G., Goldenberg C. J. Partial purification and properties of a pre-mRNA splicing activity. J Biol Chem. 1985 Jan 25;260(2):1096–1102. [PubMed] [Google Scholar]
  6. Frendewey D., Keller W. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences. Cell. 1985 Aug;42(1):355–367. doi: 10.1016/s0092-8674(85)80131-8. [DOI] [PubMed] [Google Scholar]
  7. Garrett-Wheeler E., Lockard R. E., Kumar A. Mapping of psoralen cross-linked nucleotides in RNA. Nucleic Acids Res. 1984 Apr 11;12(7):3405–3423. doi: 10.1093/nar/12.7.3405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goldenberg C. J., Hauser S. D. Accurate and efficient in vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome. Nucleic Acids Res. 1983 Mar 11;11(5):1337–1348. doi: 10.1093/nar/11.5.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goldenberg C. J. In vitro-synthesized adenovirus 2 messenger RNA precursors are accurately spliced by nuclear extracts. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4707–4711. doi: 10.1073/pnas.81.15.4707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grabowski P. J., Padgett R. A., Sharp P. A. Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate. Cell. 1984 Jun;37(2):415–427. doi: 10.1016/0092-8674(84)90372-6. [DOI] [PubMed] [Google Scholar]
  11. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Grifo J. A., Abramson R. D., Satler C. A., Merrick W. C. RNA-stimulated ATPase activity of eukaryotic initiation factors. J Biol Chem. 1984 Jul 10;259(13):8648–8654. [PubMed] [Google Scholar]
  14. Isaacs S. T., Shen C. K., Hearst J. E., Rapoport H. Synthesis and characterization of new psoralen derivatives with superior photoreactivity with DNA and RNA. Biochemistry. 1977 Mar 22;16(6):1058–1064. doi: 10.1021/bi00625a005. [DOI] [PubMed] [Google Scholar]
  15. Jacobson A. B., Good L., Simonetti J., Zuker M. Some simple computational methods to improve the folding of large RNAs. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):45–52. doi: 10.1093/nar/12.1part1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kaltwasser G., Spitzer S. G., Goldenberg C. J. Assembly in an in vitro splicing reaction of a mouse insulin messenger RNA precursor into a 60-40S ribonucleoprotein complex. Nucleic Acids Res. 1986 May 12;14(9):3687–3701. doi: 10.1093/nar/14.9.3687. [DOI] [PMC free article] [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. Krämer A., Keller W., Appel B., Lührmann R. The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors. Cell. 1984 Aug;38(1):299–307. doi: 10.1016/0092-8674(84)90551-8. [DOI] [PubMed] [Google Scholar]
  19. Kühne T., Wieringa B., Reiser J., Weissmann C. Evidence against a scanning model of RNA splicing. EMBO J. 1983;2(5):727–733. doi: 10.1002/j.1460-2075.1983.tb01492.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lang K. M., Spritz R. A. RNA splice site selection: evidence for a 5' leads to 3' scanning model. Science. 1983 Jun 24;220(4604):1351–1355. doi: 10.1126/science.6304877. [DOI] [PubMed] [Google Scholar]
  21. Mount S. M., Pettersson I., Hinterberger M., Karmas A., Steitz J. A. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell. 1983 Jun;33(2):509–518. doi: 10.1016/0092-8674(83)90432-4. [DOI] [PubMed] [Google Scholar]
  22. Padgett R. A., Grabowski P. J., Konarska M. M., Seiler S., Sharp P. A. Splicing of messenger RNA precursors. Annu Rev Biochem. 1986;55:1119–1150. doi: 10.1146/annurev.bi.55.070186.005351. [DOI] [PubMed] [Google Scholar]
  23. Padgett R. A., Konarska M. M., Aebi M., Hornig H., Weissmann C., Sharp P. A. Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8349–8353. doi: 10.1073/pnas.82.24.8349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Qu H. L., Michot B., Bachellerie J. P. Improved methods for structure probing in large RNAs: a rapid 'heterologous' sequencing approach is coupled to the direct mapping of nuclease accessible sites. Application to the 5' terminal domain of eukaryotic 28S rRNA. Nucleic Acids Res. 1983 Sep 10;11(17):5903–5920. doi: 10.1093/nar/11.17.5903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ray B. K., Lawson T. G., Kramer J. C., Cladaras M. H., Grifo J. A., Abramson R. D., Merrick W. C., Thach R. E. ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors. J Biol Chem. 1985 Jun 25;260(12):7651–7658. [PubMed] [Google Scholar]
  26. Rebagliati M. R., Melton D. A. Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity. Cell. 1987 Feb 27;48(4):599–605. doi: 10.1016/0092-8674(87)90238-8. [DOI] [PubMed] [Google Scholar]
  27. Reed R., Maniatis T. A role for exon sequences and splice-site proximity in splice-site selection. Cell. 1986 Aug 29;46(5):681–690. doi: 10.1016/0092-8674(86)90343-0. [DOI] [PubMed] [Google Scholar]
  28. Rogers J. H. The origin and evolution of retroposons. Int Rev Cytol. 1985;93:187–279. doi: 10.1016/s0074-7696(08)61375-3. [DOI] [PubMed] [Google Scholar]
  29. Ruskin B., Green M. R. Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing. Cell. 1985 Nov;43(1):131–142. doi: 10.1016/0092-8674(85)90018-2. [DOI] [PubMed] [Google Scholar]
  30. Solnick D. Alternative splicing caused by RNA secondary structure. Cell. 1985 Dec;43(3 Pt 2):667–676. doi: 10.1016/0092-8674(85)90239-9. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Wieringa B., Hofer E., Weissmann C. A minimal intron length but no specific internal sequence is required for splicing the large rabbit beta-globin intron. Cell. 1984 Jul;37(3):915–925. doi: 10.1016/0092-8674(84)90426-4. [DOI] [PubMed] [Google Scholar]
  33. Wollenzien P. L., Cantor C. R. Gel electrophoretic technique for separating crosslinked RNAs. Application to improved electron microscopic analysis of psoralen crosslinked 16 S ribosomal RNA. J Mol Biol. 1982 Jul 25;159(1):151–166. doi: 10.1016/0022-2836(82)90036-5. [DOI] [PubMed] [Google Scholar]
  34. Wollenzien P., Hearst J. E., Thammana P., Cantor C. R. Base-pairing between distant regions of the Escherichia coli 16 S ribosomal RNA in solution. J Mol Biol. 1979 Nov 25;135(1):255–269. doi: 10.1016/0022-2836(79)90351-6. [DOI] [PubMed] [Google Scholar]
  35. Youvan D. C., Hearst J. E. Sequencing psoralen photochemically reactive sites in Escherichia coli 16 S rRNA. Anal Biochem. 1982 Jan 1;119(1):86–89. doi: 10.1016/0003-2697(82)90669-8. [DOI] [PubMed] [Google Scholar]
  36. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES