Abstract
AU-rich elements (AREs) located in the 3' UTRs of the messenger RNAs (mRNAs) of many mammalian early response genes promote rapid mRNA turnover. HuR, an RRM-containing RNA-binding protein, specifically interacts with AREs, stabilizing these mRNAs. HuR is primarily nucleoplasmic, but shuttles between the nucleus and the cytoplasm via a domain called HNS located between RRM2 and RRM3. We recently showed that HuR interacts with two protein ligands, pp32 and APRIL, which are also shuttling proteins, but rely on NES domains recognized by CRM1 for export. Here we show that heat shock induces increased association of HuR with pp32 and APRIL through protein-protein interactions and that these ligands partially colocalize with HuR in cytoplasmic foci. HuR associations with the hnRNP complex also increase, but through RNA links. CRM1 coimmunoprecipitates with HuR only after heat shock, and nuclear export of HuR becomes sensitive to leptomycin B, an inhibitor of CRM1. Export after heat shock requires the same domains of HuR (HNS and RRM3) that are essential for binding pp32 and APRIL. In situ hybridization and coimmunoprecipitation experiments show that LMB treatment blocks both hsp70 mRNA nuclear export and its cytoplasmic interaction with HuR after heat shock. Together, our results argue that upon heat shock, HuR switches its export pathway to that of its ligands pp32 and APRIL, which involves the nuclear export factor CRM1. HuR and its ligands may be instrumental in the nuclear export of heat-shock mRNAs.
Full Text
The Full Text of this article is available as a PDF (6.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Akamatsu W., Okano H. J., Osumi N., Inoue T., Nakamura S., Sakakibara S., Miura M., Matsuo N., Darnell R. B., Okano H. Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and the peripheral nervous systems. Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9885–9890. doi: 10.1073/pnas.96.17.9885. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Andrews G. K., Harding M. A., Calvet J. P., Adamson E. D. The heat shock response in HeLa cells is accompanied by elevated expression of the c-fos proto-oncogene. Mol Cell Biol. 1987 Oct;7(10):3452–3458. doi: 10.1128/mcb.7.10.3452. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Antic D., Keene J. D. Embryonic lethal abnormal visual RNA-binding proteins involved in growth, differentiation, and posttranscriptional gene expression. Am J Hum Genet. 1997 Aug;61(2):273–278. doi: 10.1086/514866. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Banerji S. S., Berg L., Morimoto R. I. Transcription and post-transcriptional regulation of avian HSP70 gene expression. J Biol Chem. 1986 Nov 25;261(33):15740–15745. [PubMed] [Google Scholar]
- Blaxall B. C., Pellett A. C., Wu S. C., Pende A., Port J. D. Purification and characterization of beta-adrenergic receptor mRNA-binding proteins. J Biol Chem. 2000 Feb 11;275(6):4290–4297. doi: 10.1074/jbc.275.6.4290. [DOI] [PubMed] [Google Scholar]
- Brennan C. M., Gallouzi I. E., Steitz J. A. Protein ligands to HuR modulate its interaction with target mRNAs in vivo. J Cell Biol. 2000 Oct 2;151(1):1–14. doi: 10.1083/jcb.151.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brennan C. M., Steitz J. A. HuR and mRNA stability. Cell Mol Life Sci. 2001 Feb;58(2):266–277. doi: 10.1007/PL00000854. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Campos A. R., Grossman D., White K. Mutant alleles at the locus elav in Drosophila melanogaster lead to nervous system defects. A developmental-genetic analysis. J Neurogenet. 1985 Jun;2(3):197–218. doi: 10.3109/01677068509100150. [DOI] [PubMed] [Google Scholar]
- Chen C. Y., Shyu A. B. AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem Sci. 1995 Nov;20(11):465–470. doi: 10.1016/s0968-0004(00)89102-1. [DOI] [PubMed] [Google Scholar]
- Chen T. H., Brody J. R., Romantsev F. E., Yu J. G., Kayler A. E., Voneiff E., Kuhajda F. P., Pasternack G. R. Structure of pp32, an acidic nuclear protein which inhibits oncogene-induced formation of transformed foci. Mol Biol Cell. 1996 Dec;7(12):2045–2056. doi: 10.1091/mbc.7.12.2045. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Choi Y. D., Dreyfuss G. Monoclonal antibody characterization of the C proteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells. J Cell Biol. 1984 Dec;99(6):1997–1204. doi: 10.1083/jcb.99.6.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DeMaria C. T., Brewer G. AUF1 binding affinity to A+U-rich elements correlates with rapid mRNA degradation. J Biol Chem. 1996 May 24;271(21):12179–12184. doi: 10.1074/jbc.271.21.12179. [DOI] [PubMed] [Google Scholar]
- Ducret C., Maira S. M., Dierich A., Wasylyk B. The net repressor is regulated by nuclear export in response to anisomycin, UV, and heat shock. Mol Cell Biol. 1999 Oct;19(10):7076–7087. doi: 10.1128/mcb.19.10.7076. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fan X. C., Steitz J. A. HNS, a nuclear-cytoplasmic shuttling sequence in HuR. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15293–15298. doi: 10.1073/pnas.95.26.15293. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fan X. C., Steitz J. A. Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs. EMBO J. 1998 Jun 15;17(12):3448–3460. doi: 10.1093/emboj/17.12.3448. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fornerod M., Ohno M., Yoshida M., Mattaj I. W. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell. 1997 Sep 19;90(6):1051–1060. doi: 10.1016/s0092-8674(00)80371-2. [DOI] [PubMed] [Google Scholar]
- Forrester W., Stutz F., Rosbash M., Wickens M. Defects in mRNA 3'-end formation, transcription initiation, and mRNA transport associated with the yeast mutation prp20: possible coupling of mRNA processing and chromatin structure. Genes Dev. 1992 Oct;6(10):1914–1926. doi: 10.1101/gad.6.10.1914. [DOI] [PubMed] [Google Scholar]
- Fujita M., Kawano T., Ohta A., Sakamoto H. Neuronal expression of a Caenorhabditis elegans elav-like gene and the effect of its ectopic expression. Biochem Biophys Res Commun. 1999 Jul 14;260(3):646–652. doi: 10.1006/bbrc.1999.0957. [DOI] [PubMed] [Google Scholar]
- Fukuda M., Asano S., Nakamura T., Adachi M., Yoshida M., Yanagida M., Nishida E. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature. 1997 Nov 20;390(6657):308–311. doi: 10.1038/36894. [DOI] [PubMed] [Google Scholar]
- Gallouzi I. E., Brennan C. M., Stenberg M. G., Swanson M. S., Eversole A., Maizels N., Steitz J. A. HuR binding to cytoplasmic mRNA is perturbed by heat shock. Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3073–3078. doi: 10.1073/pnas.97.7.3073. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Good P. J. A conserved family of elav-like genes in vertebrates. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4557–4561. doi: 10.1073/pnas.92.10.4557. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gorospe M., Wang X., Holbrook N. J. p53-dependent elevation of p21Waf1 expression by UV light is mediated through mRNA stabilization and involves a vanadate-sensitive regulatory system. Mol Cell Biol. 1998 Mar;18(3):1400–1407. doi: 10.1128/mcb.18.3.1400. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gueydan C., Droogmans L., Chalon P., Huez G., Caput D., Kruys V. Identification of TIAR as a protein binding to the translational regulatory AU-rich element of tumor necrosis factor alpha mRNA. J Biol Chem. 1999 Jan 22;274(4):2322–2326. doi: 10.1074/jbc.274.4.2322. [DOI] [PubMed] [Google Scholar]
- Görlich D., Kutay U. Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol. 1999;15:607–660. doi: 10.1146/annurev.cellbio.15.1.607. [DOI] [PubMed] [Google Scholar]
- Hirsch H. H., Nair A. P., Backenstoss V., Moroni C. Interleukin-3 mRNA stabilization by a trans-acting mechanism in autocrine tumors lacking interleukin-3 gene rearrangements. J Biol Chem. 1995 Sep 1;270(35):20629–20635. doi: 10.1074/jbc.270.35.20629. [DOI] [PubMed] [Google Scholar]
- Ho J. H., Kallstrom G., Johnson A. W. Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit. J Cell Biol. 2000 Nov 27;151(5):1057–1066. doi: 10.1083/jcb.151.5.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hong Y., Sarge K. D. Regulation of protein phosphatase 2A activity by heat shock transcription factor 2. J Biol Chem. 1999 May 7;274(19):12967–12970. doi: 10.1074/jbc.274.19.12967. [DOI] [PubMed] [Google Scholar]
- 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]
- Jacobson A., Peltz S. W. Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu Rev Biochem. 1996;65:693–739. doi: 10.1146/annurev.bi.65.070196.003401. [DOI] [PubMed] [Google Scholar]
- Janssens V., Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J. 2001 Feb 1;353(Pt 3):417–439. doi: 10.1042/0264-6021:3530417. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jensen T. H., Patricio K., McCarthy T., Rosbash M. A block to mRNA nuclear export in S. cerevisiae leads to hyperadenylation of transcripts that accumulate at the site of transcription. Mol Cell. 2001 Apr;7(4):887–898. doi: 10.1016/s1097-2765(01)00232-5. [DOI] [PubMed] [Google Scholar]
- Kasashima K., Terashima K., Yamamoto K., Sakashita E., Sakamoto H. Cytoplasmic localization is required for the mammalian ELAV-like protein HuD to induce neuronal differentiation. Genes Cells. 1999 Nov;4(11):667–683. doi: 10.1046/j.1365-2443.1999.00292.x. [DOI] [PubMed] [Google Scholar]
- Kedersha N. L., Gupta M., Li W., Miller I., Anderson P. RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules. J Cell Biol. 1999 Dec 27;147(7):1431–1442. doi: 10.1083/jcb.147.7.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kedersha N., Cho M. R., Li W., Yacono P. W., Chen S., Gilks N., Golan D. E., Anderson P. Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules. J Cell Biol. 2000 Dec 11;151(6):1257–1268. doi: 10.1083/jcb.151.6.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Komiyama K., Okada K., Tomisaka S., Umezawa I., Hamamoto T., Beppu T. Antitumor activity of leptomycin B. J Antibiot (Tokyo) 1985 Mar;38(3):427–429. doi: 10.7164/antibiotics.38.427. [DOI] [PubMed] [Google Scholar]
- Kudo N., Matsumori N., Taoka H., Fujiwara D., Schreiner E. P., Wolff B., Yoshida M., Horinouchi S. Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9112–9117. doi: 10.1073/pnas.96.16.9112. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lagnado C. A., Brown C. Y., Goodall G. J. AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A). Mol Cell Biol. 1994 Dec;14(12):7984–7995. doi: 10.1128/mcb.14.12.7984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee W. M., Lin C., Curran T. Activation of the transforming potential of the human fos proto-oncogene requires message stabilization and results in increased amounts of partially modified fos protein. Mol Cell Biol. 1988 Dec;8(12):5521–5527. doi: 10.1128/mcb.8.12.5521. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lerner E. A., Lerner M. R., Janeway C. A., Jr, Steitz J. A. Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease. Proc Natl Acad Sci U S A. 1981 May;78(5):2737–2741. doi: 10.1073/pnas.78.5.2737. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Levy N. S., Chung S., Furneaux H., Levy A. P. Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR. J Biol Chem. 1998 Mar 13;273(11):6417–6423. doi: 10.1074/jbc.273.11.6417. [DOI] [PubMed] [Google Scholar]
- Li M., Makkinje A., Damuni Z. Molecular identification of I1PP2A, a novel potent heat-stable inhibitor protein of protein phosphatase 2A. Biochemistry. 1996 Jun 4;35(22):6998–7002. doi: 10.1021/bi960581y. [DOI] [PubMed] [Google Scholar]
- Liu Y., Liang S., Tartakoff A. M. Heat shock disassembles the nucleolus and inhibits nuclear protein import and poly(A)+ RNA export. EMBO J. 1996 Dec 2;15(23):6750–6757. [PMC free article] [PubMed] [Google Scholar]
- Ma W. J., Cheng S., Campbell C., Wright A., Furneaux H. Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein. J Biol Chem. 1996 Apr 5;271(14):8144–8151. doi: 10.1074/jbc.271.14.8144. [DOI] [PubMed] [Google Scholar]
- Ma W. J., Chung S., Furneaux H. The Elav-like proteins bind to AU-rich elements and to the poly(A) tail of mRNA. Nucleic Acids Res. 1997 Sep 15;25(18):3564–3569. doi: 10.1093/nar/25.18.3564. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mattaj I. W., Englmeier L. Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem. 1998;67:265–306. doi: 10.1146/annurev.biochem.67.1.265. [DOI] [PubMed] [Google Scholar]
- Mencinger M., Panagopoulos I., Contreras J. A., Mitelman F., Aman P. Expression analysis and chromosomal mapping of a novel human gene, APRIL, encoding an acidic protein rich in leucines. Biochim Biophys Acta. 1998 Jan 21;1395(2):176–180. doi: 10.1016/s0167-4781(97)00165-6. [DOI] [PubMed] [Google Scholar]
- Michael W. M., Choi M., Dreyfuss G. A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell. 1995 Nov 3;83(3):415–422. doi: 10.1016/0092-8674(95)90119-1. [DOI] [PubMed] [Google Scholar]
- Nakielny S., Dreyfuss G. The hnRNP C proteins contain a nuclear retention sequence that can override nuclear export signals. J Cell Biol. 1996 Sep;134(6):1365–1373. doi: 10.1083/jcb.134.6.1365. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakielny S., Dreyfuss G. Transport of proteins and RNAs in and out of the nucleus. Cell. 1999 Dec 23;99(7):677–690. doi: 10.1016/s0092-8674(00)81666-9. [DOI] [PubMed] [Google Scholar]
- Neville M., Stutz F., Lee L., Davis L. I., Rosbash M. The importin-beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export. Curr Biol. 1997 Oct 1;7(10):767–775. doi: 10.1016/s0960-9822(06)00335-6. [DOI] [PubMed] [Google Scholar]
- Ohno M., Segref A., Bachi A., Wilm M., Mattaj I. W. PHAX, a mediator of U snRNA nuclear export whose activity is regulated by phosphorylation. Cell. 2000 Apr 14;101(2):187–198. doi: 10.1016/S0092-8674(00)80829-6. [DOI] [PubMed] [Google Scholar]
- Okano H. J., Darnell R. B. A hierarchy of Hu RNA binding proteins in developing and adult neurons. J Neurosci. 1997 May 1;17(9):3024–3037. doi: 10.1523/JNEUROSCI.17-09-03024.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peng S. S., Chen C. Y., Xu N., Shyu A. B. RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein. EMBO J. 1998 Jun 15;17(12):3461–3470. doi: 10.1093/emboj/17.12.3461. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Piecyk M., Wax S., Beck A. R., Kedersha N., Gupta M., Maritim B., Chen S., Gueydan C., Kruys V., Streuli M. TIA-1 is a translational silencer that selectively regulates the expression of TNF-alpha. EMBO J. 2000 Aug 1;19(15):4154–4163. doi: 10.1093/emboj/19.15.4154. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Piñol-Roma S., Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature. 1992 Feb 20;355(6362):730–732. doi: 10.1038/355730a0. [DOI] [PubMed] [Google Scholar]
- Saavedra C. A., Hammell C. M., Heath C. V., Cole C. N. Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p. Genes Dev. 1997 Nov 1;11(21):2845–2856. doi: 10.1101/gad.11.21.2845. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saavedra C., Tung K. S., Amberg D. C., Hopper A. K., Cole C. N. Regulation of mRNA export in response to stress in Saccharomyces cerevisiae. Genes Dev. 1996 Jul 1;10(13):1608–1620. doi: 10.1101/gad.10.13.1608. [DOI] [PubMed] [Google Scholar]
- Sadis S., Hickey E., Weber L. A. Effect of heat shock on RNA metabolism in HeLa cells. J Cell Physiol. 1988 Jun;135(3):377–386. doi: 10.1002/jcp.1041350304. [DOI] [PubMed] [Google Scholar]
- Saito S., Miyaji-Yamaguchi M., Shimoyama T., Nagata K. Functional domains of template-activating factor-I as a protein phosphatase 2A inhibitor. Biochem Biophys Res Commun. 1999 Jun 7;259(2):471–475. doi: 10.1006/bbrc.1999.0790. [DOI] [PubMed] [Google Scholar]
- Siomi M. C., Eder P. S., Kataoka N., Wan L., Liu Q., Dreyfuss G. Transportin-mediated nuclear import of heterogeneous nuclear RNP proteins. J Cell Biol. 1997 Sep 22;138(6):1181–1192. doi: 10.1083/jcb.138.6.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith P. R., Williams D. G., Venables P. J., Maini R. N. Monoclonal antibodies to the Sjögren's syndrome associated antigen SS-B (La). J Immunol Methods. 1985 Feb 28;77(1):63–76. doi: 10.1016/0022-1759(85)90184-x. [DOI] [PubMed] [Google Scholar]
- Stutz F., Neville M., Rosbash M. Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast. Cell. 1995 Aug 11;82(3):495–506. doi: 10.1016/0092-8674(95)90438-7. [DOI] [PubMed] [Google Scholar]
- Tian Q., Streuli M., Saito H., Schlossman S. F., Anderson P. A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induces DNA fragmentation in target cells. Cell. 1991 Nov 1;67(3):629–639. doi: 10.1016/0092-8674(91)90536-8. [DOI] [PubMed] [Google Scholar]
- Vainberg I. E., Dower K., Rosbash M. Nuclear export of heat shock and non-heat-shock mRNA occurs via similar pathways. Mol Cell Biol. 2000 Jun;20(11):3996–4005. doi: 10.1128/mcb.20.11.3996-4005.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang W., Furneaux H., Cheng H., Caldwell M. C., Hutter D., Liu Y., Holbrook N., Gorospe M. HuR regulates p21 mRNA stabilization by UV light. Mol Cell Biol. 2000 Feb;20(3):760–769. doi: 10.1128/mcb.20.3.760-769.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- van der Houven van Oordt W., Diaz-Meco M. T., Lozano J., Krainer A. R., Moscat J., Cáceres J. F. The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation. J Cell Biol. 2000 Apr 17;149(2):307–316. doi: 10.1083/jcb.149.2.307. [DOI] [PMC free article] [PubMed] [Google Scholar]