Abstract
In higher eukaryotes, the regulation of pre-mRNA processing is still poorly known. The accumulation of various mature mRNAs, which can be observed in the nuclei of mammalian cells, is suggestive of a regulatory role of transport. However, the significance of these nuclear mRNA is presently unknown. We have used a tetracycline-regulated promoter to investigate the dynamics of these pools of mRNAs upon arrest of transcription. We observed, for beta-globin and LT-alpha genes, a slow disappearance of these mRNA from the nucleus, with an apparent half-life that is similar to their cytoplasmic half-life. In view of these dynamics, these mRNA cannot simply be mature mRNAs in transit to the cytoplasm. They could be mRNAs retained in the nucleus, provided that the regulation of mRNA stability is comparable in the nucleus and the cytoplasm. But, because of their limited stability, these nuclear mRNAs cannot constitute a significant stock for gene expression. Alternatively, they could reflect a bidirectional transport of mRNA, that is, to and from the cytoplasm, which would provide a direct explanation for the similarity in both compartments of their half-life and poly(A) tail shortening over time.
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- Allmang C., Petfalski E., Podtelejnikov A., Mann M., Tollervey D., Mitchell P. The yeast exosome and human PM-Scl are related complexes of 3' --> 5' exonucleases. Genes Dev. 1999 Aug 15;13(16):2148–2158. doi: 10.1101/gad.13.16.2148. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bassell G. J., Powers C. M., Taneja K. L., Singer R. H. Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts. J Cell Biol. 1994 Aug;126(4):863–876. doi: 10.1083/jcb.126.4.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Belgrader P., Cheng J., Maquat L. E. Evidence to implicate translation by ribosomes in the mechanism by which nonsense codons reduce the nuclear level of human triosephosphate isomerase mRNA. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):482–486. doi: 10.1073/pnas.90.2.482. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carter M. S., Li S., Wilkinson M. F. A splicing-dependent regulatory mechanism that detects translation signals. EMBO J. 1996 Nov 1;15(21):5965–5975. [PMC free article] [PubMed] [Google Scholar]
- Ciejek E. M., Nordstrom J. L., Tsai M. J., O'Malley B. W. Ribonucleic acid precursors are associated with the chick oviduct nuclear matrix. Biochemistry. 1982 Sep 28;21(20):4945–4953. doi: 10.1021/bi00263a018. [DOI] [PubMed] [Google Scholar]
- Cramer P., Pesce C. G., Baralle F. E., Kornblihtt A. R. Functional association between promoter structure and transcript alternative splicing. Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11456–11460. doi: 10.1073/pnas.94.21.11456. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Daneholt B. A look at messenger RNP moving through the nuclear pore. Cell. 1997 Mar 7;88(5):585–588. doi: 10.1016/s0092-8674(00)81900-5. [DOI] [PubMed] [Google Scholar]
- Dirks W., Schaper F., Kirchhoff S., Morelle C., Hauser H. A multifunctional vector family for gene expression in mammalian cells. Gene. 1994 Nov 18;149(2):387–388. doi: 10.1016/0378-1119(94)90186-4. [DOI] [PubMed] [Google Scholar]
- Frey M. R., Bailey A. D., Weiner A. M., Matera A. G. Association of snRNA genes with coiled bodies is mediated by nascent snRNA transcripts. Curr Biol. 1999 Feb 11;9(3):126–135. doi: 10.1016/s0960-9822(99)80066-9. [DOI] [PubMed] [Google Scholar]
- Gondran P., Amiot F., Weil D., Dautry F. Accumulation of mature mRNA in the nuclear fraction of mammalian cells. FEBS Lett. 1999 Sep 24;458(3):324–328. doi: 10.1016/s0014-5793(99)01175-8. [DOI] [PubMed] [Google Scholar]
- Gossen M., Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547–5551. doi: 10.1073/pnas.89.12.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haaf T., Ward D. C. Inhibition of RNA polymerase II transcription causes chromatin decondensation, loss of nucleolar structure, and dispersion of chromosomal domains. Exp Cell Res. 1996 Apr 10;224(1):163–173. doi: 10.1006/excr.1996.0124. [DOI] [PubMed] [Google Scholar]
- Huang S., Deerinck T. J., Ellisman M. H., Spector D. L. In vivo analysis of the stability and transport of nuclear poly(A)+ RNA. J Cell Biol. 1994 Aug;126(4):877–899. doi: 10.1083/jcb.126.4.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Izaurralde E., Adam S. Transport of macromolecules between the nucleus and the cytoplasm. RNA. 1998 Apr;4(4):351–364. [PMC free article] [PubMed] [Google Scholar]
- Jarmolowski A., Boelens W. C., Izaurralde E., Mattaj I. W. Nuclear export of different classes of RNA is mediated by specific factors. J Cell Biol. 1994 Mar;124(5):627–635. doi: 10.1083/jcb.124.5.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loflin P., Chen C. Y., Shyu A. B. Unraveling a cytoplasmic role for hnRNP D in the in vivo mRNA destabilization directed by the AU-rich element. Genes Dev. 1999 Jul 15;13(14):1884–1897. doi: 10.1101/gad.13.14.1884. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Long B. H., Huang C. Y., Pogo A. O. Isolation and characterization of the nuclear matrix in Friend erythroleukemia cells: chromatin and hnRNA interactions with the nuclear matrix. Cell. 1979 Dec;18(4):1079–1090. doi: 10.1016/0092-8674(79)90221-6. [DOI] [PubMed] [Google Scholar]
- 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]
- Mercer J. F., Wake S. A. An analysis of the rate of metallothionein mRNA poly(A)-shortening using RNA blot hybridization. Nucleic Acids Res. 1985 Nov 25;13(22):7929–7943. doi: 10.1093/nar/13.22.7929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michael W. M. Nucleocytoplasmic shuttling signals: two for the price of one. Trends Cell Biol. 2000 Feb;10(2):46–50. doi: 10.1016/s0962-8924(99)01695-5. [DOI] [PubMed] [Google Scholar]
- Miralles F., Ofverstedt L. G., Sabri N., Aissouni Y., Hellman U., Skoglund U., Visa N. Electron tomography reveals posttranscriptional binding of pre-mRNPs to specific fibers in the nucleoplasm. J Cell Biol. 2000 Jan 24;148(2):271–282. doi: 10.1083/jcb.148.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mortillaro M. J., Blencowe B. J., Wei X., Nakayasu H., Du L., Warren S. L., Sharp P. A., Berezney R. A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8253–8257. doi: 10.1073/pnas.93.16.8253. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neel H., Gondran P., Weil D., Dautry F. Regulation of pre-mRNA processing by src. Curr Biol. 1995 Apr 1;5(4):413–422. doi: 10.1016/s0960-9822(95)00082-0. [DOI] [PubMed] [Google Scholar]
- O'Neill R. E., Jaskunas R., Blobel G., Palese P., Moroianu J. Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import. J Biol Chem. 1995 Sep 29;270(39):22701–22704. doi: 10.1074/jbc.270.39.22701. [DOI] [PubMed] [Google Scholar]
- Penman S., Vesco C., Penman M. Localization and kinetics of formation of nuclear heterodisperse RNA, cytoplasmic heterodisperse RNA and polyribosome-associated messenger RNA in HeLa cells. J Mol Biol. 1968 May 28;34(1):49–60. doi: 10.1016/0022-2836(68)90234-9. [DOI] [PubMed] [Google Scholar]
- Riedel N., Bachmann M., Prochnow D., Richter H. P., Fasold H. Permeability measurements with closed vesicles from rat liver nuclear envelopes. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3540–3544. doi: 10.1073/pnas.84.11.3540. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ross D. A., Yen R. W., Chae C. B. Association of globin ribonucleic acid and its precursors with the chicken erythroblast nuclear matrix. Biochemistry. 1982 Feb 16;21(4):764–771. doi: 10.1021/bi00533a029. [DOI] [PubMed] [Google Scholar]
- Schiavi S. C., Wellington C. L., Shyu A. B., Chen C. Y., Greenberg M. E., Belasco J. G. Multiple elements in the c-fos protein-coding region facilitate mRNA deadenylation and decay by a mechanism coupled to translation. J Biol Chem. 1994 Feb 4;269(5):3441–3448. [PubMed] [Google Scholar]
- Schröder H. C., Facy P., Monsigny M., Pfeifer K., Bek A., Müller W. E. Purification of a glucose-binding protein from rat liver nuclei. Evidence for a role in targeting of nuclear mRNP to nuclear pore complex. Eur J Biochem. 1992 May 1;205(3):1017–1025. doi: 10.1111/j.1432-1033.1992.tb16869.x. [DOI] [PubMed] [Google Scholar]
- Schröder H. C., Rottmann M., Bachmann M., Müller W. E., McDonald A. R., Agutter P. S. Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system. Eur J Biochem. 1986 Aug 15;159(1):51–59. doi: 10.1111/j.1432-1033.1986.tb09832.x. [DOI] [PubMed] [Google Scholar]
- Schumm D. E., Tejwani R., Webb T. E. Hybridization analysis of RNA transported from rat liver nuclei in response to 35 kDa normal and 60 kDa messenger RNA transport factors. Biochim Biophys Acta. 1989 Sep 21;1009(1):54–60. doi: 10.1016/0167-4781(89)90078-x. [DOI] [PubMed] [Google Scholar]
- Singh O. P., Björkroth B., Masich S., Wieslander L., Daneholt B. The intranuclear movement of Balbiani ring premessenger ribonucleoprotein particles. Exp Cell Res. 1999 Aug 25;251(1):135–146. doi: 10.1006/excr.1999.4490. [DOI] [PubMed] [Google Scholar]
- Stutz F., Rosbash M. Nuclear RNA export. Genes Dev. 1998 Nov 1;12(21):3303–3319. doi: 10.1101/gad.12.21.3303. [DOI] [PubMed] [Google Scholar]
- Taneja K. L., Lifshitz L. M., Fay F. S., Singer R. H. Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy. J Cell Biol. 1992 Dec;119(5):1245–1260. doi: 10.1083/jcb.119.5.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thermann R., Neu-Yilik G., Deters A., Frede U., Wehr K., Hagemeier C., Hentze M. W., Kulozik A. E. Binary specification of nonsense codons by splicing and cytoplasmic translation. EMBO J. 1998 Jun 15;17(12):3484–3494. doi: 10.1093/emboj/17.12.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Veyrune J. L., Campbell G. P., Wiseman J., Blanchard J. M., Hesketh J. E. A localisation signal in the 3' untranslated region of c-myc mRNA targets c-myc mRNA and beta-globin reporter sequences to the perinuclear cytoplasm and cytoskeletal-bound polysomes. J Cell Sci. 1996 Jun;109(Pt 6):1185–1194. doi: 10.1242/jcs.109.6.1185. [DOI] [PubMed] [Google Scholar]
- Visa N., Izaurralde E., Ferreira J., Daneholt B., Mattaj I. W. A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export. J Cell Biol. 1996 Apr;133(1):5–14. doi: 10.1083/jcb.133.1.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weil D., Brosset S., Dautry F. RNA processing is a limiting step for murine tumor necrosis factor beta expression in response to interleukin-2. Mol Cell Biol. 1990 Nov;10(11):5865–5875. doi: 10.1128/mcb.10.11.5865. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xu N., Loflin P., Chen C. Y., Shyu A. B. A broader role for AU-rich element-mediated mRNA turnover revealed by a new transcriptional pulse strategy. Nucleic Acids Res. 1998 Jan 15;26(2):558–565. doi: 10.1093/nar/26.2.558. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yuryev A., Patturajan M., Litingtung Y., Joshi R. V., Gentile C., Gebara M., Corden J. L. The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):6975–6980. doi: 10.1073/pnas.93.14.6975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang W., Wagner B. J., Ehrenman K., Schaefer A. W., DeMaria C. T., Crater D., DeHaven K., Long L., Brewer G. Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1. Mol Cell Biol. 1993 Dec;13(12):7652–7665. doi: 10.1128/mcb.13.12.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]