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. 1998 Feb 1;26(3):721–729. doi: 10.1093/nar/26.3.721

Efficient 3'-end formation of human beta-globin mRNA in vivo requires sequences within the last intron but occurs independently of the splicing reaction.

M Antoniou 1, F Geraghty 1, J Hurst 1, F Grosveld 1
PMCID: PMC147328  PMID: 9443963

Abstract

The second intron (betaIVS-II) of the human beta-globin gene is essential for the accumulation of stable cytoplasmic mRNA and is implicated in promoting efficient 3'-end formation. This report presents quantitative comparisons between betaIVS-II mutants at physiological levels of expression from within a natural chromatin context in vivo which further defines it's function. In marked contrast to a beta-globin gene lacking a second intron, two mutants defective in splicing (small size or a splice donor mutation), still undergo essentially normal levels of 3'-end formation and in the absence of exon skipping. Therefore, 3' cleavage of beta-globin transcripts requires the presence of betaIVS-II sequences, but not the splicing reaction. The placement of betaIVS-II in the IVS-I position did not reduce the efficiency of 3' cleavage indicating that the distance between the necessary element(s) in this intron and the polyadenylation recognition site is not a crucial factor. Subsequent placement of betaIVS-I in the intron II position, reduced the efficiency of 3'-end formation to only 16% of normal. A direct replacement of intron II by the heterologous introns betaIVS-I or alpha-globin IVS-II, only partially substitute (16 and 30% respectively) for betaIVS-II. Hybrid introns show that efficient 3'-end formation is strongly enhanced by the presence of the terminal 60 nt of betaIVS-II. These data imply that the last intervening sequence of multiple intron containing genes is a principal determinant of the efficiency of 3'-end formation and may act as a post-transcriptional regulatory step in gene expression.

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Selected References

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  1. Antoniou M., deBoer E., Habets G., Grosveld F. The human beta-globin gene contains multiple regulatory regions: identification of one promoter and two downstream enhancers. EMBO J. 1988 Feb;7(2):377–384. doi: 10.1002/j.1460-2075.1988.tb02824.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ash J., Ke Y., Korb M., Johnson L. F. Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene. Mol Cell Biol. 1993 Mar;13(3):1565–1571. doi: 10.1128/mcb.13.3.1565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Behringer R. R., Hammer R. E., Brinster R. L., Palmiter R. D., Townes T. M. Two 3' sequences direct adult erythroid-specific expression of human beta-globin genes in transgenic mice. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7056–7060. doi: 10.1073/pnas.84.20.7056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boelens W. C., Jansen E. J., van Venrooij W. J., Stripecke R., Mattaj I. W., Gunderson S. I. The human U1 snRNP-specific U1A protein inhibits polyadenylation of its own pre-mRNA. Cell. 1993 Mar 26;72(6):881–892. doi: 10.1016/0092-8674(93)90577-d. [DOI] [PubMed] [Google Scholar]
  5. Brinster R. L., Allen J. M., Behringer R. R., Gelinas R. E., Palmiter R. D. Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci U S A. 1988 Feb;85(3):836–840. doi: 10.1073/pnas.85.3.836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buchman A. R., Berg P. Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol. 1988 Oct;8(10):4395–4405. doi: 10.1128/mcb.8.10.4395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collis P., Antoniou M., Grosveld F. Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression. EMBO J. 1990 Jan;9(1):233–240. doi: 10.1002/j.1460-2075.1990.tb08100.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Du L., Warren S. L. A functional interaction between the carboxy-terminal domain of RNA polymerase II and pre-mRNA splicing. J Cell Biol. 1997 Jan 13;136(1):5–18. doi: 10.1083/jcb.136.1.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gasser C. S., Simonsen C. C., Schilling J. W., Schimke R. T. Expression of abbreviated mouse dihydrofolate reductase genes in cultured hamster cells. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6522–6526. doi: 10.1073/pnas.79.21.6522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Green M. R. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. doi: 10.1146/annurev.cb.07.110191.003015. [DOI] [PubMed] [Google Scholar]
  11. Gruss P., Lai C. J., Dhar R., Khoury G. Splicing as a requirement for biogenesis of functional 16S mRNA of simian virus 40. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4317–4321. doi: 10.1073/pnas.76.9.4317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gunderson S. I., Beyer K., Martin G., Keller W., Boelens W. C., Mattaj L. W. The human U1A snRNP protein regulates polyadenylation via a direct interaction with poly(A) polymerase. Cell. 1994 Feb 11;76(3):531–541. doi: 10.1016/0092-8674(94)90116-3. [DOI] [PubMed] [Google Scholar]
  13. Hamer D. H., Leder P. Splicing and the formation of stable RNA. Cell. 1979 Dec;18(4):1299–1302. doi: 10.1016/0092-8674(79)90240-x. [DOI] [PubMed] [Google Scholar]
  14. Horowitz D. S., Krainer A. R. Mechanisms for selecting 5' splice sites in mammalian pre-mRNA splicing. Trends Genet. 1994 Mar;10(3):100–106. doi: 10.1016/0168-9525(94)90233-x. [DOI] [PubMed] [Google Scholar]
  15. Huang M. T., Gorman C. M. Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 1990 Feb 25;18(4):937–947. doi: 10.1093/nar/18.4.937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jonsson J. J., Foresman M. D., Wilson N., McIvor R. S. Intron requirement for expression of the human purine nucleoside phosphorylase gene. Nucleic Acids Res. 1992 Jun 25;20(12):3191–3198. doi: 10.1093/nar/20.12.3191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kim E., Du L., Bregman D. B., Warren S. L. Splicing factors associate with hyperphosphorylated RNA polymerase II in the absence of pre-mRNA. J Cell Biol. 1997 Jan 13;136(1):19–28. doi: 10.1083/jcb.136.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liu X., Mertz J. E. HnRNP L binds a cis-acting RNA sequence element that enables intron-dependent gene expression. Genes Dev. 1995 Jul 15;9(14):1766–1780. doi: 10.1101/gad.9.14.1766. [DOI] [PubMed] [Google Scholar]
  19. Liu X., Mertz J. E. Sequence of the polypyrimidine tract of the 3'-terminal 3' splicing signal can affect intron-dependent pre-mRNA processing in vivo. Nucleic Acids Res. 1996 May 1;24(9):1765–1773. doi: 10.1093/nar/24.9.1765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lou H., Gagel R. F., Berget S. M. An intron enhancer recognized by splicing factors activates polyadenylation. Genes Dev. 1996 Jan 15;10(2):208–219. doi: 10.1101/gad.10.2.208. [DOI] [PubMed] [Google Scholar]
  21. Lou H., Yang Y., Cote G. J., Berget S. M., Gagel R. F. An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene. Mol Cell Biol. 1995 Dec;15(12):7135–7142. doi: 10.1128/mcb.15.12.7135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lutz C. S., Alwine J. C. Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal. Genes Dev. 1994 Mar 1;8(5):576–586. doi: 10.1101/gad.8.5.576. [DOI] [PubMed] [Google Scholar]
  23. Lutz C. S., Murthy K. G., Schek N., O'Connor J. P., Manley J. L., Alwine J. C. Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro. Genes Dev. 1996 Feb 1;10(3):325–337. doi: 10.1101/gad.10.3.325. [DOI] [PubMed] [Google Scholar]
  24. McCracken S., Fong N., Yankulov K., Ballantyne S., Pan G., Greenblatt J., Patterson S. D., Wickens M., Bentley D. L. The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature. 1997 Jan 23;385(6614):357–361. doi: 10.1038/385357a0. [DOI] [PubMed] [Google Scholar]
  25. Moreira A., Wollerton M., Monks J., Proudfoot N. J. Upstream sequence elements enhance poly(A) site efficiency of the C2 complement gene and are phylogenetically conserved. EMBO J. 1995 Aug 1;14(15):3809–3819. doi: 10.1002/j.1460-2075.1995.tb00050.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nesic D., Cheng J., Maquat L. E. Sequences within the last intron function in RNA 3'-end formation in cultured cells. Mol Cell Biol. 1993 Jun;13(6):3359–3369. doi: 10.1128/mcb.13.6.3359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nesic D., Zhang J., Maquat L. E. Lack of an effect of the efficiency of RNA 3'-end formation on the efficiency of removal of either the final or the penultimate intron in intact cells. Mol Cell Biol. 1995 Jan;15(1):488–496. doi: 10.1128/mcb.15.1.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Neuberger M. S., Williams G. T. The intron requirement for immunoglobulin gene expression is dependent upon the promoter. Nucleic Acids Res. 1988 Jul 25;16(14B):6713–6724. doi: 10.1093/nar/16.14.6713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Niwa M., Berget S. M. Mutation of the AAUAAA polyadenylation signal depresses in vitro splicing of proximal but not distal introns. Genes Dev. 1991 Nov;5(11):2086–2095. doi: 10.1101/gad.5.11.2086. [DOI] [PubMed] [Google Scholar]
  30. Niwa M., Rose S. D., Berget S. M. In vitro polyadenylation is stimulated by the presence of an upstream intron. Genes Dev. 1990 Sep;4(9):1552–1559. doi: 10.1101/gad.4.9.1552. [DOI] [PubMed] [Google Scholar]
  31. Palmiter R. D., Sandgren E. P., Avarbock M. R., Allen D. D., Brinster R. L. Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):478–482. doi: 10.1073/pnas.88.2.478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Proudfoot N. Ending the message is not so simple. Cell. 1996 Nov 29;87(5):779–781. doi: 10.1016/s0092-8674(00)81982-0. [DOI] [PubMed] [Google Scholar]
  33. Proudfoot N. Poly(A) signals. Cell. 1991 Feb 22;64(4):671–674. doi: 10.1016/0092-8674(91)90495-k. [DOI] [PubMed] [Google Scholar]
  34. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  35. Steinmetz E. J. Pre-mRNA processing and the CTD of RNA polymerase II: the tail that wags the dog? Cell. 1997 May 16;89(4):491–494. doi: 10.1016/s0092-8674(00)80230-5. [DOI] [PubMed] [Google Scholar]
  36. Wahle E. 3'-end cleavage and polyadenylation of mRNA precursors. Biochim Biophys Acta. 1995 Apr 4;1261(2):183–194. doi: 10.1016/0167-4781(94)00248-2. [DOI] [PubMed] [Google Scholar]
  37. Wahle E., Keller W. The biochemistry of 3'-end cleavage and polyadenylation of messenger RNA precursors. Annu Rev Biochem. 1992;61:419–440. doi: 10.1146/annurev.bi.61.070192.002223. [DOI] [PubMed] [Google Scholar]
  38. Wall L., deBoer E., Grosveld F. The human beta-globin gene 3' enhancer contains multiple binding sites for an erythroid-specific protein. Genes Dev. 1988 Sep;2(9):1089–1100. doi: 10.1101/gad.2.9.1089. [DOI] [PubMed] [Google Scholar]
  39. Wassarman K. M., Steitz J. A. Association with terminal exons in pre-mRNAs: a new role for the U1 snRNP? Genes Dev. 1993 Apr;7(4):647–659. doi: 10.1101/gad.7.4.647. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. deBoer E., Antoniou M., Mignotte V., Wall L., Grosveld F. The human beta-globin promoter; nuclear protein factors and erythroid specific induction of transcription. EMBO J. 1988 Dec 20;7(13):4203–4212. doi: 10.1002/j.1460-2075.1988.tb03317.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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