Journal of Experimental Botany, Vol. 53, No. 378, pp. 2277-2278,
November 1, 2002
© 2002 Oxford University Press
Cloning and characterization of Arabidopsis homologues of the animal CstF complex that regulates 3' mRNA cleavage and polyadenylation
Received 11 June 2002; Accepted 3 July 2002
Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel
1 To whom correspondence should be addressed. Fax: +972 8 9344181. E-mail: gad.galili{at}weizmann.ac.il
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Abstract |
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The 3' cleavage and polyadenylation of mRNAs has been studied in detail in animals and yeast, but not in plants. Aimed at elucidating the regulation of mRNA 3' end formation in plants, three Arabidopsis cDNAs encoding homologues of the animal proteins CstF-64, CstF-77 and CstF-50 that form the cleavage stimulating factor of the polyadenylation machinery have been cloned. It is shown experimentally that the N-terminal domain of the Arabidopsis CstF-64 homologue binds the mRNA 3' non-coding region in an analogous manner to the animal protein. It is also shown that the Arabidopsis CstF-64 and CstF-77 homologues strongly interact with each other in a similar way to their animal counterparts. These results imply that these Arabidopsis homologues belong to the polyadenylation machinery of nuclear mRNAs.
Key words: Key words: Arabidopsis, CstF complex, mRNA cleavage and polyadenylation, regulation.
The 3' end processing of nuclear mRNAs in animal cells includes both cleavage and polyadenylation and is regulated by several multi-subunit complexes (Zhao et al., 1999). One of these complexes is the cleavage stimulation factor (CstF), consisting of three subunits termed CstF-77, CstF-64 and CstF-50 (Zhao et al., 1999). The CstF-64 subunit of this complex binds, via its N-terminal domain, a U-rich sequence that is generally situated downstream from the cleavage/polyadenylation site and also interacts with CstF-77 (Zhao et al., 1999). In turn, CstF-77 interacts with a second complex, the cleavage polyadenylation specificity factor (CSPF), which binds the consensus AAUAAA polyadenylation signal (Zhao et al., 1999). Plant mRNAs contain analogous sequences in similar positions to the animal AAUAAA and U-rich sequences (Graber et al., 1999), suggesting an equivalent regulatory mechanisms in plants, but the machinery of mRNA 3' end formation in plants is still unknown.
Three Arabidopsis cDNA homologues of the animal CstF-64 (AtCstF-64) (GenBank Accession No. AF515695), CstF-77 (AtCstF-64) (GenBank Accession No. AF515697) and CstF-50 (AtCstF-64) (GenBank Accession No. AF515696) proteins have been isolated. The deduced protein encoded by AtCstF-64 exhibits 32% identity and 44% similarity to the human CstF-64. The proteins encoded by AtCstF-77 and the human CstF-77 exhibit 32% identity and 49% similarity in their amino acid sequences. The protein encoded by AtCstF-50 exhibits 37% identity and 55% similarity to the human CstF-50.
To test whether the N-terminal 110 amino acids of the AtCstF-64 homologue can bind RNA, this domain was produced, fused to a six histidines (His) tag, in Escherichia coli, using the pQE31 expression vector (Qiagen, Hilden, Germany). Using the T7 promoter of pBluescript (Stratagene, CA, USA), a 191 nt long 32[P]-labelled RNA derived from the 3' terminator region of the octopine synthetase gene of Agrobacterium tumefaciens was also transcribed in vitro. As shown in Fig. 1, mixing of increasing molar amounts of the AtCstF-64 N-terminal domain with the octopine synthetase 3 terminator RNA resulted in progressively slower migration of the 32[P]-radiolabelled band in a native polyacrylamide gel. This could result either from interaction of the N-terminal domain of AtCstF-64 with several different regions of the octopine synthetase 3 terminator RNA or from multimerization of the protein, in a concentration-dependent manner. Similar observations were reported using the N-terminal domain of the human CstF-64 subunit (Takagaki and Manley, 1997).
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To test for potential interactions between the AtCstF-64 and AtCstF-77 gene products, their open reading frames were subcloned downstream to a SP6 promoter in the plasmid pTNT (Promega, WI, USA). A DNA fragment encoding a haemagglutinin (HA) epitope tag was also fused in frame immediately downstream from the initiator ATG codon of AtCstF-77. These plasmids were used to transcribe mRNAs, which were then translated in a rabbit reticulocyte lysate in the presence of 35S-methionine. As shown in Fig. 2, a protein product with the expected size of AtCstF-64 was synthesized from AtCstF-64 and was immunoprecipitated with polyclonal anti-AtCstF-64 antibodies, but not in the absence of these antibodies (Fig. 2A, lanes a, b). In reactions containing AtCstF-77 as a template, two major polypeptides were synthesized, which were immunoprecipitated by the monoclonal anti-HA antibody (Covance, VA, USA), but not in the absence of this antibody (Fig. 2B, lanes a, b). These polypeptides were also within the expected size range for AtCstF-77 and apparently represented both the full-length protein and a truncated polypeptide resulting from an early translation termination. When the AtCstF-64 and AtCstF-77 polypeptides were mixed, both were co-immunoprecipitated with either the anti-AtCstF-64 or the anti-HA tag antibodies (lane d in Fig. 2A and B, respectively). This co-immunoprecipitation did not result from cross reactivities of the antibodies since the anti-AtCstF-64 and the anti-HA antibodies did not cross-react with the AtCstF-77 and AtCstF-64 polypeptides, respectively (lane c in Fig. 2A and B, respectively).
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Using a similar expression system, a His-tagged protein from AtCstF-50 was also synthesized and its potential interaction with AtCstF-77, as occurs in animals (Zhao et al., 1999), was tested. No interaction between these two proteins was observed in this experimental system (data not shown).
Taken together, the results suggest that the Arabidopsis proteins encoded by AtCstF-64 and AtCstF-77 belong to an RNA binding complex that functions in a similar way to the animal CstF in mRNA 3' end formation. Whether the protein encoded by AtCstF-50 also belongs to this complex still awaits further confirmation.
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Acknowledgements |
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We thank Dr Avihai Danon for his help with the experiment described in Fig. 1. This work was supported by the Israel Academy of Sciences and Humanities, National Council for Research and Development, Israel. GG is an incumbent of the Bronfman Chair of Plant Sciences.
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Graber JH, Cantor CR, Mohr SC, Smith TF. 1999. In silico detection of control signals: mRNA 3'-end-processing sequences in diverse species. Proceedings of the National Academy of Sciences, USA 96, 14055–14060.
Grafi G, Burnett RJ, Helentjaris T, Larkins BA, DeCaprio JA, Sellers WR, Kaelin Jr WG. 1996. A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication. Proceedings of the National Academy of Sciences, USA 93, 8962–8967.
Katz YS, Danon A. 2002. The 3'-untranslated region of chloroplast psbA mRNA stabilizes binding of regulatory proteins to the leader of the message. Journal of Biological Chemistry 277, 18665–18669.
Takagaki Y, Manley JL. 1997. RNA recognition by the human polyadenylation factor CstF. Molecular and Cellular Biology 17, 3907–3914.
Zhao J, Hyman L, Moore C. 1999. Formation of mRNA 3' ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiology and Molecular Biology Reviews 63, 405–445.
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