Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Nucleic Acids Research logoLink to Nucleic Acids Research
. 1990 Feb 25;18(4):937–947. doi: 10.1093/nar/18.4.937

Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.

M T Huang 1, C M Gorman 1
PMCID: PMC330348  PMID: 1690394

Abstract

Two expression vectors were constructed that differ only in the presence (+) or absence (-) of an intervening sequence (IVS) in their 5'-untranslated leaders. Transient transfection into four mammalian cell lines resulted in higher levels of the indicator protein (CAT) from the IVS(+) vector (6 to 50-fold). Cytoplasmic RNA concentrations in 293s and HeLa cell lines corresponded directly to resultant protein levels; measurements in 293s cells of transcription initiation and elongation, steady-state total nuclear RNA, and cytoplasmic RNA stability, were equivalent for the two vectors. Surprisingly, the amount of poly(A)+ nuclear RNA was greater from the IVS(+) vector. Since this difference matches the ratio seen with polyadenylated cytoplasmic RNA, our results imply that splicing is coupled to a polyadenylation/transport pathway.

Full text

PDF
937

Images in this article

Selected References

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

  1. Babich A., Feldman L. T., Nevins J. R., Darnell J. E., Jr, Weinberger C. Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination. Mol Cell Biol. 1983 Jul;3(7):1212–1221. doi: 10.1128/mcb.3.7.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banerji J., Olson L., Schaffner W. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell. 1983 Jul;33(3):729–740. doi: 10.1016/0092-8674(83)90015-6. [DOI] [PubMed] [Google Scholar]
  3. Berget S. M., Moore C., Sharp P. A. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3171–3175. doi: 10.1073/pnas.74.8.3171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnstiel M. L., Busslinger M., Strub K. Transcription termination and 3' processing: the end is in site! Cell. 1985 Jun;41(2):349–359. doi: 10.1016/s0092-8674(85)80007-6. [DOI] [PubMed] [Google Scholar]
  5. Boggs R. T., Gregor P., Idriss S., Belote J. M., McKeown M. Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene. Cell. 1987 Aug 28;50(5):739–747. doi: 10.1016/0092-8674(87)90332-1. [DOI] [PubMed] [Google Scholar]
  6. Bothwell A. L., Paskind M., Reth M., Imanishi-Kari T., Rajewsky K., Baltimore D. Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a gamma 2a variable region. Cell. 1981 Jun;24(3):625–637. doi: 10.1016/0092-8674(81)90089-1. [DOI] [PubMed] [Google Scholar]
  7. Breitbart R. E., Nadal-Ginard B. Developmentally induced, muscle-specific trans factors control the differential splicing of alternative and constitutive troponin T exons. Cell. 1987 Jun 19;49(6):793–803. doi: 10.1016/0092-8674(87)90617-9. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Callis J., Fromm M., Walbot V. Introns increase gene expression in cultured maize cells. Genes Dev. 1987 Dec;1(10):1183–1200. doi: 10.1101/gad.1.10.1183. [DOI] [PubMed] [Google Scholar]
  11. Campos R., Villarreal L. P. An SV40 deletion mutant accumulates late transcripts in a paranuclear extract. Virology. 1982 May;119(1):1–11. doi: 10.1016/0042-6822(82)90059-9. [DOI] [PubMed] [Google Scholar]
  12. Carlock L., Jones N. C. Synthesis of an unspliced cytoplasmic message by an adenovirus 5 deletion mutant. Nature. 1981 Dec 10;294(5841):572–574. doi: 10.1038/294572a0. [DOI] [PubMed] [Google Scholar]
  13. Chow L. T., Gelinas R. E., Broker T. R., Roberts R. J. An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA. Cell. 1977 Sep;12(1):1–8. doi: 10.1016/0092-8674(77)90180-5. [DOI] [PubMed] [Google Scholar]
  14. Drayna D., Fielding C., McLean J., Baer B., Castro G., Chen E., Comstock L., Henzel W., Kohr W., Rhee L. Cloning and expression of human apolipoprotein D cDNA. J Biol Chem. 1986 Dec 15;261(35):16535–16539. [PubMed] [Google Scholar]
  15. 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]
  16. Ghosh P. K., Roy P., Barkan A., Mertz J. E., Weissman S. M., Lebowitz P. Unspliced functional late 19S mRNAs containing intervening sequences are produced by a late leader mutant of simian virus 40. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1386–1390. doi: 10.1073/pnas.78.3.1386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gillies S. D., Morrison S. L., Oi V. T., Tonegawa S. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell. 1983 Jul;33(3):717–728. doi: 10.1016/0092-8674(83)90014-4. [DOI] [PubMed] [Google Scholar]
  18. Gilmartin G. M., McDevitt M. A., Nevins J. R. Multiple factors are required for specific RNA cleavage at a poly(A) addition site. Genes Dev. 1988 May;2(5):578–587. doi: 10.1101/gad.2.5.578. [DOI] [PubMed] [Google Scholar]
  19. Gorman C. M., Gies D., McCray G., Huang M. The human cytomegalovirus major immediate early promoter can be trans-activated by adenovirus early proteins. Virology. 1989 Aug;171(2):377–385. doi: 10.1016/0042-6822(89)90605-3. [DOI] [PubMed] [Google Scholar]
  20. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gorman C., Padmanabhan R., Howard B. H. High efficiency DNA-mediated transformation of primate cells. Science. 1983 Aug 5;221(4610):551–553. doi: 10.1126/science.6306768. [DOI] [PubMed] [Google Scholar]
  22. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  23. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  24. Gross M. K., Kainz M. S., Merrill G. F. Introns are inconsequential to efficient formation of cellular thymidine kinase mRNA in mouse L cells. Mol Cell Biol. 1987 Dec;7(12):4576–4581. doi: 10.1128/mcb.7.12.4576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gruss P., Khoury G. Rescue of a splicing defective mutant by insertion of an heterologous intron. Nature. 1980 Aug 7;286(5773):634–637. doi: 10.1038/286634a0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Hunt C., Morimoto R. I. Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6455–6459. doi: 10.1073/pnas.82.19.6455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kao H. T., Nevins J. R. Transcriptional activation and subsequent control of the human heat shock gene during adenovirus infection. Mol Cell Biol. 1983 Nov;3(11):2058–2065. doi: 10.1128/mcb.3.11.2058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kedes L. H. Histone genes and histone messengers. Annu Rev Biochem. 1979;48:837–870. doi: 10.1146/annurev.bi.48.070179.004201. [DOI] [PubMed] [Google Scholar]
  31. Khoury G., Gruss P., Dhar R., Lai C. J. Processing and expression of early SV40 mRNA: a role for RNA conformation in splicing. Cell. 1979 Sep;18(1):85–92. doi: 10.1016/0092-8674(79)90356-8. [DOI] [PubMed] [Google Scholar]
  32. Lai C. J., Khoury G. Deletion mutants of simian virus 40 defective in biosynthesis of late viral mRNA. Proc Natl Acad Sci U S A. 1979 Jan;76(1):71–75. doi: 10.1073/pnas.76.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Laski F. A., Rio D. C., Rubin G. M. Tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing. Cell. 1986 Jan 17;44(1):7–19. doi: 10.1016/0092-8674(86)90480-0. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Linial M., Gunderson N., Groudine M. Enhanced transcription of c-myc in bursal lymphoma cells requires continuous protein synthesis. Science. 1985 Dec 6;230(4730):1126–1132. doi: 10.1126/science.2999973. [DOI] [PubMed] [Google Scholar]
  36. Mansour S. L., Grodzicker T., Tjian R. Downstream sequences affect transcription initiation from the adenovirus major late promoter. Mol Cell Biol. 1986 Jul;6(7):2684–2694. doi: 10.1128/mcb.6.7.2684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Moore C. L., Chen J., Whoriskey J. Two proteins crosslinked to RNA containing the adenovirus L3 poly(A) site require the AAUAAA sequence for binding. EMBO J. 1988 Oct;7(10):3159–3169. doi: 10.1002/j.1460-2075.1988.tb03183.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Moore C. L., Sharp P. A. Accurate cleavage and polyadenylation of exogenous RNA substrate. Cell. 1985 Jul;41(3):845–855. doi: 10.1016/s0092-8674(85)80065-9. [DOI] [PubMed] [Google Scholar]
  39. Moore C. L., Sharp P. A. Site-specific polyadenylation in a cell-free reaction. Cell. 1984 Mar;36(3):581–591. doi: 10.1016/0092-8674(84)90337-4. [DOI] [PubMed] [Google Scholar]
  40. Mory Y. Y., Gefter M. L. Synthesis of messenger RNA-like molecules in isolated myeloma nuclei. Nucleic Acids Res. 1977 Jun;4(6):1739–1757. doi: 10.1093/nar/4.6.1739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mowry K. L., Steitz J. A. Identification of the human U7 snRNP as one of several factors involved in the 3' end maturation of histone premessenger RNA's. Science. 1987 Dec 18;238(4834):1682–1687. doi: 10.1126/science.2825355. [DOI] [PubMed] [Google Scholar]
  42. Mulligan R. C., Berg P. Expression of a bacterial gene in mammalian cells. Science. 1980 Sep 19;209(4463):1422–1427. doi: 10.1126/science.6251549. [DOI] [PubMed] [Google Scholar]
  43. Mulligan R. C., Howard B. H., Berg P. Synthesis of rabbit beta-globin in cultured monkey kidney cells following infection with a SV40 beta-globin recombinant genome. Nature. 1979 Jan 11;277(5692):108–114. doi: 10.1038/277108a0. [DOI] [PubMed] [Google Scholar]
  44. Nordeen S. K., Green P. P., 3rd, Fowlkes D. M. A rapid, sensitive, and inexpensive assay for chloramphenicol acetyltransferase. DNA. 1987 Apr;6(2):173–178. doi: 10.1089/dna.1987.6.173. [DOI] [PubMed] [Google Scholar]
  45. Reddy V. B., Ghosh P. K., Lebowitz P., Piatak M., Weissman S. M. Simian virus 40 early mRNA's. I. Genomic localization of 3' and 5' termini and two major splices in mRNA from transformed and lytically infected cells. J Virol. 1979 Apr;30(1):279–296. doi: 10.1128/jvi.30.1.279-296.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Sternberg E. A., Spizz G., Perry W. M., Vizard D., Weil T., Olson E. N. Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene. Mol Cell Biol. 1988 Jul;8(7):2896–2909. doi: 10.1128/mcb.8.7.2896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Thomas P. S. Hybridization of denatured RNA transferred or dotted nitrocellulose paper. Methods Enzymol. 1983;100:255–266. doi: 10.1016/0076-6879(83)00060-9. [DOI] [PubMed] [Google Scholar]
  49. Treisman R., Novak U., Favaloro J., Kamen R. Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature. 1981 Aug 13;292(5824):595–600. doi: 10.1038/292595a0. [DOI] [PubMed] [Google Scholar]
  50. Villarreal L. P., Carr S. Genetic test for involvement of intervening sequences in transport of nuclear RNA. Mol Cell Biol. 1982 Dec;2(12):1550–1557. doi: 10.1128/mcb.2.12.1550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Villarreal L. P., White R. T. A splice junction deletion deficient in the transport of RNA does not polyadenylate nuclear RNA. Mol Cell Biol. 1983 Aug;3(8):1381–1388. doi: 10.1128/mcb.3.8.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wilusz J., Feig D. I., Shenk T. The C proteins of heterogeneous nuclear ribonucleoprotein complexes interact with RNA sequences downstream of polyadenylation cleavage sites. Mol Cell Biol. 1988 Oct;8(10):4477–4483. doi: 10.1128/mcb.8.10.4477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wu B., Hunt C., Morimoto R. Structure and expression of the human gene encoding major heat shock protein HSP70. Mol Cell Biol. 1985 Feb;5(2):330–341. doi: 10.1128/mcb.5.2.330. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES