Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1989 Oct 25;17(20):8159–8169. doi: 10.1093/nar/17.20.8159

A secondary structure at the 3' splice site affects the in vitro splicing reaction of mouse immunoglobulin mu chain pre-mRNAs.

A Watakabe 1, K Inoue 1, H Sakamoto 1, Y Shimura 1
PMCID: PMC334955  PMID: 2510128

Abstract

The expression of the IgM (immunoglobulin mu) heavy chain gene is known to be regulated at the post-transcriptional level. The two isoforms, the membrane-bound and secreted forms, are generated from the same gene by alternative processing at the 3' end of the primary transcript. The processing reactions involved are polyadenylation at the upstream poly(A) site (for the secreted form) and polyadenylation at the downstream poly(A) site coupled with splicing between exon C4 and exon M1 (for the membrane-bound form). The regulatory mechanism underlying these differential processing reactions is still not well understood. We investigated the splicing reaction between exon C4 and exon M1 in a HeLa nuclear extract using model transcripts containing the 5' and 3' splice sites of the C4-M1 intron. We found that the 3' splice site of the C4-M1 intron is sequestered in a stem-loop structure, which inhibits the splicing reaction in vitro. The inhibition by the stem-loop structure was also observed with a mouse lymphoma extract.

Full text

PDF
8159

Images in this article

Selected References

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

  1. Alt F. W., Bothwell A. L., Knapp M., Siden E., Mather E., Koshland M., Baltimore D. Synthesis of secreted and membrane-bound immunoglobulin mu heavy chains is directed by mRNAs that differ at their 3' ends. Cell. 1980 Jun;20(2):293–301. doi: 10.1016/0092-8674(80)90615-7. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Early P., Rogers J., Davis M., Calame K., Bond M., Wall R., Hood L. Two mRNAs can be produced from a single immunoglobulin mu gene by alternative RNA processing pathways. Cell. 1980 Jun;20(2):313–319. doi: 10.1016/0092-8674(80)90617-0. [DOI] [PubMed] [Google Scholar]
  4. England T. E., Bruce A. G., Uhlenbeck O. C. Specific labeling of 3' termini of RNA with T4 RNA ligase. Methods Enzymol. 1980;65(1):65–74. doi: 10.1016/s0076-6879(80)65011-3. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Fu X. Y., Manley J. L. Factors influencing alternative splice site utilization in vivo. Mol Cell Biol. 1987 Feb;7(2):738–748. doi: 10.1128/mcb.7.2.738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Galli G., Guise J. W., McDevitt M. A., Tucker P. W., Nevins J. R. Relative position and strengths of poly(A) sites as well as transcription termination are critical to membrane versus secreted mu-chain expression during B-cell development. Genes Dev. 1987 Jul;1(5):471–481. doi: 10.1101/gad.1.5.471. [DOI] [PubMed] [Google Scholar]
  9. Galli G., Guise J., Tucker P. W., Nevins J. R. Poly(A) site choice rather than splice site choice governs the regulated production of IgM heavy-chain RNAs. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2439–2443. doi: 10.1073/pnas.85.8.2439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gorski K., Carneiro M., Schibler U. Tissue-specific in vitro transcription from the mouse albumin promoter. Cell. 1986 Dec 5;47(5):767–776. doi: 10.1016/0092-8674(86)90519-2. [DOI] [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. Kelley D. E., Perry R. P. Transcriptional and posttranscriptional control of immunoglobulin mRNA production during B lymphocyte development. Nucleic Acids Res. 1986 Jul 11;14(13):5431–5447. doi: 10.1093/nar/14.13.5431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Konarska M. M., Padgett R. A., Sharp P. A. Recognition of cap structure in splicing in vitro of mRNA precursors. Cell. 1984 Oct;38(3):731–736. doi: 10.1016/0092-8674(84)90268-x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Leff S. E., Rosenfeld M. G., Evans R. M. Complex transcriptional units: diversity in gene expression by alternative RNA processing. Annu Rev Biochem. 1986;55:1091–1117. doi: 10.1146/annurev.bi.55.070186.005303. [DOI] [PubMed] [Google Scholar]
  16. Milstein C. P., Deverson E. V., Rabbitts T. H. The sequence of the human immunoglobulin mu-delta intron reveals possible vestigial switch segments. Nucleic Acids Res. 1984 Aug 24;12(16):6523–6535. doi: 10.1093/nar/12.16.6523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nevins J. R. The pathway of eukaryotic mRNA formation. Annu Rev Biochem. 1983;52:441–466. doi: 10.1146/annurev.bi.52.070183.002301. [DOI] [PubMed] [Google Scholar]
  18. Ohno M., Sakamoto H., Shimura Y. Preferential excision of the 5' proximal intron from mRNA precursors with two introns as mediated by the cap structure. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5187–5191. doi: 10.1073/pnas.84.15.5187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Peterson M. L., Perry R. P. Regulated production of mu m and mu s mRNA requires linkage of the poly(A) addition sites and is dependent on the length of the mu s-mu m intron. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8883–8887. doi: 10.1073/pnas.83.23.8883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Peterson M. L., Perry R. P. The regulated production of mu m and mu s mRNA is dependent on the relative efficiencies of mu s poly(A) site usage and the c mu 4-to-M1 splice. Mol Cell Biol. 1989 Feb;9(2):726–738. doi: 10.1128/mcb.9.2.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rogers J., Early P., Carter C., Calame K., Bond M., Hood L., Wall R. Two mRNAs with different 3' ends encode membrane-bound and secreted forms of immunoglobulin mu chain. Cell. 1980 Jun;20(2):303–312. doi: 10.1016/0092-8674(80)90616-9. [DOI] [PubMed] [Google Scholar]
  23. Sakamoto H., Ohno M., Yasuda K., Mizumoto K., Shimura Y. In vitro splicing of a chicken delta-crystallin pre-mRNA in a mammalian nuclear extract. J Biochem. 1987 Nov;102(5):1289–1301. doi: 10.1093/oxfordjournals.jbchem.a122167. [DOI] [PubMed] [Google Scholar]
  24. Sawa H., Ohno M., Sakamoto H., Shimura Y. Requirement of ATP in the second step of the pre-mRNA splicing reaction. Nucleic Acids Res. 1988 Apr 25;16(8):3157–3164. doi: 10.1093/nar/16.8.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Tsurushita N., Avdalovic N. M., Korn L. J. Regulation of differential processing of mouse immunoglobulin mu heavy-chain mRNA. Nucleic Acids Res. 1987 Jun 11;15(11):4603–4615. doi: 10.1093/nar/15.11.4603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tsurushita N., Ho L., Korn L. J. Nuclear factors in B lymphoma enhance splicing of mouse membrane-bound mu mRNA in Xenopus oocytes. Science. 1988 Jan 29;239(4839):494–497. doi: 10.1126/science.3124268. [DOI] [PubMed] [Google Scholar]
  29. Tsurushita N., Korn L. J. Effects of intron length on differential processing of mouse mu heavy-chain mRNA. Mol Cell Biol. 1987 Jul;7(7):2602–2605. doi: 10.1128/mcb.7.7.2602. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES