Abstract
In mammalian cells, the enzymatic pathways involved in cytoplasmic mRNA decay are incompletely defined. In this study, we have used two approaches to disrupt activities of deadenylating and/or decapping enzymes to monitor effects on mRNA decay kinetics and trap decay intermediates. Our results show that deadenylation is the key first step that triggers decay of both wild-type stable and nonsense codon–containing unstable β-globin mRNAs in mouse NIH3T3 fibroblasts. PAN2 and CCR4 are the major poly(A) nucleases active in cytoplasmic deadenylation that have biphasic kinetics, with PAN2 initiating deadenylation followed by CCR4-mediated poly(A) shortening. DCP2-mediated decapping takes place after deadenylation and may serve as a backup mechanism for triggering mRNA decay when initial deadenylation by PAN2 is compromised. Our findings reveal a functional link between deadenylation and decapping and help to define in vivo pathways for mammalian cytoplasmic mRNA decay.
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References
Mitchell, P. & Tollervey, D. mRNA stability in eukaryotes. Curr. Opin. Genet. Dev. 10, 193–198 (2000).
Wilusz, C.J., Wormington, M. & Peltz, S.W. The cap-to-tail guide to mRNA turnover. Nat. Rev. Mol. Cell Biol. 2, 237–246 (2001).
Fritz, D.T., Bergman, N., Kilpatrick, W.J., Wilusz, C.J. & Wilusz, J. Messenger RNA decay in mammalian cells: the exonuclease perspective. Cell Biochem. Biophys. 41, 265–278 (2004).
Meyer, S., Temme, C. & Wahle, E. Messenger RNA turnover in eukaryotes: Pathways and enzymes. Crit. Rev. Biochem. Mol. Biol. 39, 197–216 (2004).
Parker, R. & Song, H. The enzymes and control of eukaryotic mRNA turnover. Nat. Struct. Mol. Biol. 11, 121–127 (2004).
Daugeron, M.-C., Mauxion, F. & Seraphin, B. The yeast POP2 gene encodes a nuclease involved in mRNA deadenylation. Nucleic Acids Res. 29, 2448–2455 (2001).
Boeck, R. et al. The yeast Pan2 protein is required for poly(A)-binding protein-stimulated poly(A)-nuclease activity. J. Biol. Chem. 271, 432–438 (1996).
Brown, C., Tarun, S., Jr . Boeck, R. & Sachs, A. PAN3 encodes a subunit of the Pab1p-dependent poly(A) nuclease in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 5744–5753 (1996).
Tucker, M. et al. The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell 104, 377–386 (2001).
Gao, M., Fritz, D.T., Ford, L.P. & Wilusz, J. Interaction between a poly(A)-specific ribonuclease and the 5′ cap influences mRNA deadenylation rates in vitro. Mol. Cell 5, 479–488 (2000).
Dehlin, E., Wormington, M., Korner, C.G. & Wahle, E. Cap-dependent deadenylation of mRNA. EMBO J. 19, 1079–1086 (2000).
Körner, C.G. & Wahle, E. Poly(A) tail shortening by a mammalian poly(A)-specific 3′-exoribonuclease. J. Biol. Chem. 272, 10448–10456 (1997).
Muhlrad, D. & Parker, R. Premature translational termination triggers mRNA decapping. Nature 370, 578–581 (1994).
Cao, D. & Parker, R. Computational modeling and experimental analysis of nonsense-mediated decay in yeast. Cell 113, 533–545 (2003).
Mitchell, P. & Tollervey, D. An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3′ → 5′ degradation. Mol. Cell 11, 1405–1413 (2003).
Chen, C.-Y.A. & Shyu, A.-B. Rapid deadenylation triggered by a nonsense codon precedes decay of the RNA body in a mammalian cytoplasmic nonsense-mediated decay pathway. Mol. Cell. Biol. 23, 4805–4813 (2003).
Couttet, P. & Grange, T. Premature termination codons enhance mRNA decapping in human cells. Nucleic Acids Res. 32, 488–494 (2004).
Chang, T.-C. et al. UNR, a new partner of poly(A)-binding protein, plays a key role in translationally coupled mRNA turnover mediated by the c-fos major coding-region determinant. Genes Dev. 18, 2010–2023 (2004).
Shyu, A.-B., Greenberg, M.E. & Belasco, J.G. The c-fos mRNA is targeted for rapid decay by two distinct mRNA degradation pathways. Genes Dev. 3, 60–72 (1989).
Winzen, R. et al. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J. 18, 4969–4980 (1999).
Xu, N., Loflin, P., Chen, C.-Y.A. & Shyu, A.-B. A broader role for AU-rich element-mediated mRNA turnover revealed by a new transcriptional pulse strategy. Nucleic Acids Res. 26, 558–565 (1998).
Dupressoir, A. et al. Identification of four families of yCCR4- and Mg2+-dependent endonuclease-related proteins in higher eukaryotes, and characterization of orthologs of yCCR4 with a conserved leucine-rich repeat essential for hCAF1/hPOP2 binding. BMC Genomics [online] 2, 9 (2001)(10.1186/1471-2164-2-9).
Albert, T.K. et al. Isolation and characterization of human orthologs of yeast CCR4-NOT complex subunits. Nucleic Acids Res. 28, 809–817 (2000).
Chen, C.-Y.A., Xu, N., Zhu, W. & Shyu, A.-B. Functional dissection of hnRNP D suggests that nuclear import is required before hnRNP D can modulate mRNA turnover in the cytoplasm. RNA 10, 669–680 (2004).
Shyu, A.B. & Wilkinson, M.F. The double lives of shuttling mRNA binding proteins. Cell 102, 135–138 (2000).
Kudo, N. et al. Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. USA 96, 9112–9117 (1999).
Kudo, N. et al. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 242, 540–547 (1998).
Mendell, J.T., Medghalchi, S.M., Lake, R.G., Noensie, E.N. & Dietz, H.C. Novel Upf2p orthologues suggest a functional link between translation initiation and nonsense surveillance complexes. Mol. Cell. Biol. 20, 8944–8957 (2000).
Ren, Y.-G., Martinez, J. & Virtanen, A. Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe2+-mediated cleavage. J. Biol. Chem. 277, 5982–5987 (2002).
Baggs, J.E. & Green, C.B. Nocturnin, a deadenylase in Xenopus laevis retina: A mechanism for posttranscriptional control of circadian-related mRNA. Curr. Biol. 13, 189–198 (2003).
Martinez, J., Ren, Y.-G., Nilsson, P., Ehrenberg, M. & Virtanen, A. The mRNA cap structure stimulates rate of poly(A) removal and amplifies processivity of degradation. J. Biol. Chem. 276, 27923–27929 (2001).
Loflin, P.T., Chen, C.-Y.A., Xu, N. & Shyu, A.-B. Transcriptional pulsing approaches for analysis of mRNA turnover in mammalian cells. Methods 17, 11–20 (1999).
Loflin, P.T., Chen, C.-Y.A. & Shyu, A.-B. Unraveling a cytoplasmic role for hnRNP D in the in vivo mRNA destabilization directed by the AU-rich element. Genes Dev. 13, 1884–1897 (1999).
Fischer, N. & Weis, K. The DEAD box protein Dhh1 stimulates the decapping enzyme Dcp1. EMBO J. 21, 2788–2797 (2002).
Coller, J.M., Tucker, M., Sheth, U., Valencia-Sanchez, M.A. & Parker, R. The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes. RNA 7, 1717–1727 (2001).
Cougot, N., Babajko, S. & Seraphin, B. Cytoplasmic foci are sites of mRNA decay in human cells. J. Cell Biol. 165, 31–40 (2004).
Uchida, N., Hoshino, S. & Katada, T. Identification of a human -cytoplasmic poly(A) nuclease complex stimulated by poly(A)-binding protein. J. Biol. Chem. 279, 1383–1391 (2004).
Khanna, R. & Kiledjian, M. Poly(A)-binding-protein-mediated regulation of hDcp2 decapping in vitro. EMBO J. 23, 1968–1976 (2004).
Tucker, M., Staples, R.R., Valencia-Sanchez, M.A., Muhlrad, D. & Parker, R. Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae. EMBO J. 21, 1427–1436 (2002).
Andrei, M.A. et al. A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies. RNA 11, 717–727 (2005).
Viswanathan, P., Ohn, T., Chiang, Y.-C., Chen, J. & Denis, C.L. Mouse CAF1 can function as a processive deadenylase/3'-5′-exonuclease in vitro but in yeast the deadenylase function of CAF1 is not required for mRNA poly(A) removal. J. Biol. Chem. 279, 23988–23995 (2004).
Temme, C., Zaessinger, S., Meyer, S., Simonelig, M. & Wahle, E. A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila. EMBO J. 23, 2862–2871 (2004).
Yamashita, A., Ohnishi, T., Kashima, I., Taya, Y. & Ohno, S. Human SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense-mediated mRNA decay. Genes Dev. 15, 2215–2228 (2001).
Chen, C.Y. & Shyu, A.B. Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements. Mol. Cell. Biol. 14, 8471–8482 (1994).
Shyu, A.-B., Garcia-Sanz, J.A. & Mullner, E. Analysis of mRNA decay in mammalian cells. in The Immunology Methods Manual (ed. Lefkovits, I.) 450–62 (Academic Press, London, 1996).
Shyu, A.-B., Belasco, J.G. & Greenberg, M.G. Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. Genes Dev. 5, 221–232 (1991).
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. 17, 3461–3470 (1998).
Acknowledgements
We thank R. Kulmacz, J. Lever and A. van Hoof for critical reading of the manuscript and their comments and Y. Tao for technical assistance with the heterokaryon experiments. This work was supported by a grant from the US National Institutes of Health (GM46454) to A.-B.S.
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Supplementary information
Supplementary Fig. 1
Catalytically inactive poly(A) nuclease mutants remain able to associate with their corresponding partners in vivo. (PDF 3028 kb)
Supplementary Fig. 2
Semi-log plots showing mRNA decay kinetics. (PDF 317 kb)
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Yamashita, A., Chang, TC., Yamashita, Y. et al. Concerted action of poly(A) nucleases and decapping enzyme in mammalian mRNA turnover. Nat Struct Mol Biol 12, 1054–1063 (2005). https://doi.org/10.1038/nsmb1016
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DOI: https://doi.org/10.1038/nsmb1016
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