Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1989 Nov;9(11):4852–4861. doi: 10.1128/mcb.9.11.4852

The 216-nucleotide intron of the E1A pre-mRNA contains a hairpin structure that permits utilization of unusually distant branch acceptors.

K Chebli 1, R Gattoni 1, P Schmitt 1, G Hildwein 1, J Stevenin 1
PMCID: PMC363635  PMID: 2601698

Abstract

A recently characterized 216-nucleotide intron-splicing reaction occurs within the adenovirus E1A pre-mRNA through the use of three branch acceptor sites, located at 59, 55, and 51 nucleotides from the 3' splice site. To investigate the role of the cis-acting sequence elements in the selection of such unusually distant branch sites, transcripts differing in sequence downstream of the branch sites were analyzed for in vitro splicing. Initial results suggested that secondary structure could be involved in the use of distant branch sites. The involvement of a hairpin structure, including a nine-G C-base-pair stem, was supported by the results of site-directed mutagenesis analyses. Mutations that destroyed or weakened this hairpin resulted in an inefficient splicing reaction. In contrast, complementary mutation or deletion of two bulges, which involved a restoration or reinforcement of the hairpin, resulted in a reactivation or improvement of the splicing efficiency, respectively. Therefore, we conclude that the hairpin structure shortens the operational distance between the 3' splice site and the branch acceptors and brings the branch sites into the branch-permissive window, 18 to 40 nucleotides upstream of the 3' splice site. Our results confirm the importance of the constraint of distance for the splicing reaction and show that this constraint may be overcome by means of a stable hairpin formation.

Full text

PDF
4855

Images in this article

4855Image
on p.4855
4856Image
on p.4856
4857Image
on p.4857
4858Image
on p.4858

Selected References

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

  1. Adema G. J., Bovenberg R. A., Jansz H. S., Baas P. D. Unusual branch point selection involved in splicing of the alternatively processed Calcitonin/CGRP-I pre-mRNA. Nucleic Acids Res. 1988 Oct 25;16(20):9513–9526. doi: 10.1093/nar/16.20.9513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aebi M., Hornig H., Padgett R. A., Reiser J., Weissmann C. Sequence requirements for splicing of higher eukaryotic nuclear pre-mRNA. Cell. 1986 Nov 21;47(4):555–565. doi: 10.1016/0092-8674(86)90620-3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Birchmeier C., Schümperli D., Sconzo G., Birnstiel M. L. 3' editing of mRNAs: sequence requirements and involvement of a 60-nucleotide RNA in maturation of histone mRNA precursors. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1057–1061. doi: 10.1073/pnas.81.4.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
  6. Cellini A., Felder E., Rossi J. J. Yeast pre-messenger RNA splicing efficiency depends on critical spacing requirements between the branch point and 3' splice site. EMBO J. 1986 May;5(5):1023–1030. doi: 10.1002/j.1460-2075.1986.tb04317.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chabot B., Steitz J. A. Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes. Mol Cell Biol. 1987 Jan;7(1):281–293. doi: 10.1128/mcb.7.1.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  9. Cooper T. A., Cardone M. H., Ordahl C. P. Cis requirements for alternative splicing of the cardiac troponin T pre-mRNA. Nucleic Acids Res. 1988 Sep 12;16(17):8443–8465. doi: 10.1093/nar/16.17.8443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dreyfuss G. Structure and function of nuclear and cytoplasmic ribonucleoprotein particles. Annu Rev Cell Biol. 1986;2:459–498. doi: 10.1146/annurev.cb.02.110186.002331. [DOI] [PubMed] [Google Scholar]
  11. Eperon L. P., Estibeiro J. P., Eperon I. C. The role of nucleotide sequences in splice site selection in eukaryotic pre-messenger RNA. Nature. 1986 Nov 20;324(6094):280–282. doi: 10.1038/324280a0. [DOI] [PubMed] [Google Scholar]
  12. Eperon L. P., Graham I. R., Griffiths A. D., Eperon I. C. Effects of RNA secondary structure on alternative splicing of pre-mRNA: is folding limited to a region behind the transcribing RNA polymerase? Cell. 1988 Jul 29;54(3):393–401. doi: 10.1016/0092-8674(88)90202-4. [DOI] [PubMed] [Google Scholar]
  13. Freier S. M., Kierzek R., Jaeger J. A., Sugimoto N., Caruthers M. H., Neilson T., Turner D. H. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. doi: 10.1073/pnas.83.24.9373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Freyer G. A., Arenas J., Perkins K. K., Furneaux H. M., Pick L., Young B., Roberts R. J., Hurwitz J. In vitro formation of a lariat structure containing a G2'-5'G linkage. J Biol Chem. 1987 Mar 25;262(9):4267–4273. [PubMed] [Google Scholar]
  15. Gattoni R., Schmitt P., Stevenin J. In vitro splicing of adenovirus E1A transcripts: characterization of novel reactions and of multiple branch points abnormally far from the 3' splice site. Nucleic Acids Res. 1988 Mar 25;16(6):2389–2409. doi: 10.1093/nar/16.6.2389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Green M. R., Maniatis T., Melton D. A. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell. 1983 Mar;32(3):681–694. doi: 10.1016/0092-8674(83)90054-5. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Halfter H., Gallwitz D. Impairment of yeast pre-mRNA splicing by potential secondary structure-forming sequences near the conserved branchpoint sequence. Nucleic Acids Res. 1988 Nov 25;16(22):10413–10423. doi: 10.1093/nar/16.22.10413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hall K. B., Green M. R., Redfield A. G. Structure of a pre-mRNA branch point/3' splice site region. Proc Natl Acad Sci U S A. 1988 Feb;85(3):704–708. doi: 10.1073/pnas.85.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hartmuth K., Barta A. Unusual branch point selection in processing of human growth hormone pre-mRNA. Mol Cell Biol. 1988 May;8(5):2011–2020. doi: 10.1128/mcb.8.5.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hornig H., Aebi M., Weissmann C. Effect of mutations at the lariat branch acceptor site on beta-globin pre-mRNA splicing in vitro. Nature. 1986 Dec 11;324(6097):589–591. doi: 10.1038/324589a0. [DOI] [PubMed] [Google Scholar]
  22. Keller E. B., Noon W. A. Intron splicing: a conserved internal signal in introns of animal pre-mRNAs. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7417–7420. doi: 10.1073/pnas.81.23.7417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Konarska M. M., Grabowski P. J., Padgett R. A., Sharp P. A. Characterization of the branch site in lariat RNAs produced by splicing of mRNA precursors. Nature. 1985 Feb 14;313(6003):552–557. doi: 10.1038/313552a0. [DOI] [PubMed] [Google Scholar]
  24. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  25. Krainer A. R., Maniatis T., Ruskin B., Green M. R. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. doi: 10.1016/0092-8674(84)90049-7. [DOI] [PubMed] [Google Scholar]
  26. Krämer A. Presplicing complex formation requires two proteins and U2 snRNP. Genes Dev. 1988 Sep;2(9):1155–1167. doi: 10.1101/gad.2.9.1155. [DOI] [PubMed] [Google Scholar]
  27. Langford C. J., Gallwitz D. Evidence for an intron-contained sequence required for the splicing of yeast RNA polymerase II transcripts. Cell. 1983 Jun;33(2):519–527. doi: 10.1016/0092-8674(83)90433-6. [DOI] [PubMed] [Google Scholar]
  28. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  29. Noble J. C., Pan Z. Q., Prives C., Manley J. L. Splicing of SV40 early pre-mRNA to large T and small t mRNAs utilizes different patterns of lariat branch sites. Cell. 1987 Jul 17;50(2):227–236. doi: 10.1016/0092-8674(87)90218-2. [DOI] [PubMed] [Google Scholar]
  30. Noble J. C., Prives C., Manley J. L. Alternative splicing of SV40 early pre-mRNA is determined by branch site selection. Genes Dev. 1988 Nov;2(11):1460–1475. doi: 10.1101/gad.2.11.1460. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Padgett R. A., Konarska M. M., Grabowski P. J., Hardy S. F., Sharp P. A. Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science. 1984 Aug 31;225(4665):898–903. doi: 10.1126/science.6206566. [DOI] [PubMed] [Google Scholar]
  34. Parent A., Zeitlin S., Efstratiadis A. Minimal exon sequence requirements for efficient in vitro splicing of mono-intronic nuclear pre-mRNA. J Biol Chem. 1987 Aug 15;262(23):11284–11291. [PubMed] [Google Scholar]
  35. Parker R., Siliciano P. G., Guthrie C. Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell. 1987 Apr 24;49(2):229–239. doi: 10.1016/0092-8674(87)90564-2. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Reed R., Maniatis T. Intron sequences involved in lariat formation during pre-mRNA splicing. Cell. 1985 May;41(1):95–105. doi: 10.1016/0092-8674(85)90064-9. [DOI] [PubMed] [Google Scholar]
  38. Reed R., Maniatis T. The role of the mammalian branchpoint sequence in pre-mRNA splicing. Genes Dev. 1988 Oct;2(10):1268–1276. doi: 10.1101/gad.2.10.1268. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Ruskin B., Greene J. M., Green M. R. Cryptic branch point activation allows accurate in vitro splicing of human beta-globin intron mutants. Cell. 1985 Jul;41(3):833–844. doi: 10.1016/s0092-8674(85)80064-7. [DOI] [PubMed] [Google Scholar]
  42. Ruskin B., Krainer A. R., Maniatis T., Green M. R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
  43. Ruskin B., Zamore P. D., Green M. R. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. doi: 10.1016/0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
  44. Schmitt P., Gattoni R., Keohavong P., Stévenin J. Alternative splicing of E1A transcripts of adenovirus requires appropriate ionic conditions in vitro. Cell. 1987 Jul 3;50(1):31–39. doi: 10.1016/0092-8674(87)90659-3. [DOI] [PubMed] [Google Scholar]
  45. Smith C. W., Nadal-Ginard B. Mutually exclusive splicing of alpha-tropomyosin exons enforced by an unusual lariat branch point location: implications for constitutive splicing. Cell. 1989 Mar 10;56(5):749–758. doi: 10.1016/0092-8674(89)90678-8. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Solnick D., Lee S. I. Amount of RNA secondary structure required to induce an alternative splice. Mol Cell Biol. 1987 Sep;7(9):3194–3198. doi: 10.1128/mcb.7.9.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Stephens C., Harlow E. Differential splicing yields novel adenovirus 5 E1A mRNAs that encode 30 kd and 35 kd proteins. EMBO J. 1987 Jul;6(7):2027–2035. doi: 10.1002/j.1460-2075.1987.tb02467.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Ulfendahl P. J., Linder S., Kreivi J. P., Nordqvist K., Sevensson C., Hultberg H., Akusjärvi G. A novel adenovirus-2 E1A mRNA encoding a protein with transcription activation properties. EMBO J. 1987 Jul;6(7):2037–2044. doi: 10.1002/j.1460-2075.1987.tb02468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wagner R. W., Nishikura K. Cell cycle expression of RNA duplex unwindase activity in mammalian cells. Mol Cell Biol. 1988 Feb;8(2):770–777. doi: 10.1128/mcb.8.2.770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zeitlin S., Efstratiadis A. In vivo splicing products of the rabbit beta-globin pre-mRNA. Cell. 1984 Dec;39(3 Pt 2):589–602. doi: 10.1016/0092-8674(84)90466-5. [DOI] [PubMed] [Google Scholar]
  53. Zhuang Y. A., Goldstein A. M., Weiner A. M. UACUAAC is the preferred branch site for mammalian mRNA splicing. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2752–2756. doi: 10.1073/pnas.86.8.2752. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES