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Mol Cell Biol. 1997 July; 17(7): 3907–3914.
PMCID: PMC232243

RNA recognition by the human polyadenylation factor CstF.


Polyadenylation of mammalian mRNA precursors requires at least two signal sequences in the RNA: the nearly invariant AAUAAA, situated 5' to the site of polyadenylation, and a much more variable GU- or U-rich downstream element. At least some downstream sequences are recognized by the heterotrimeric polyadenylation factor CstF, although how, and indeed if, all variations of this diffuse element are bound by a single factor is unknown. Here we show that the RNP-type RNA binding domain of the 64-kDa subunit of CstF (CstF-64) (64K RBD) is sufficient to define a functional downstream element. Selection-amplification (SELEX) experiments employing a glutathione S-transferase (GST)-64K RBD fusion protein selected GU-rich sequences that defined consensus recognition motifs closely matching those present in natural poly(A) sites. Selected sequences were bound specifically, and with surprisingly high affinities, by intact CstF and were functional in reconstituted, CstF-dependent cleavage assays. Our results also indicate that GU- and U-rich sequences are variants of a single CstF recognition motif. For comparison, SELEX was performed with a GST fusion containing the RBD from the apparent yeast homolog of CstF-64, RNA15. Strikingly, although the two RBDs are almost 50% identical and yeast poly(A) signals are at least as degenerate as the mammalian downstream element, a nearly invariant 12-base U-rich sequence distinct from the CstF-64 consensus was identified. We discuss these results in terms of the function and evolution of mRNA 3'-end signals.

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Selected References

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  • Bardwell VJ, Wickens M, Bienroth S, Keller W, Sproat BS, Lamond AI. Site-directed ribose methylation identifies 2'-OH groups in polyadenylation substrates critical for AAUAAA recognition and poly(A) addition. Cell. 1991 Apr 5;65(1):125–133. [PubMed]
  • Bienroth S, Wahle E, Suter-Crazzolara C, Keller W. Purification of the cleavage and polyadenylation factor involved in the 3'-processing of messenger RNA precursors. J Biol Chem. 1991 Oct 15;266(29):19768–19776. [PubMed]
  • Chanfreau G, Noble SM, Guthrie C. Essential yeast protein with unexpected similarity to subunits of mammalian cleavage and polyadenylation specificity factor (CPSF). Science. 1996 Nov 29;274(5292):1511–1514. [PubMed]
  • Chen J, Moore C. Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA. Mol Cell Biol. 1992 Aug;12(8):3470–3481. [PMC free article] [PubMed]
  • Chou ZF, Chen F, Wilusz J. Sequence and position requirements for uridylate-rich downstream elements of polyadenylation signals. Nucleic Acids Res. 1994 Jul 11;22(13):2525–2531. [PMC free article] [PubMed]
  • Christofori G, Keller W. 3' cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP. Cell. 1988 Sep 9;54(6):875–889. [PubMed]
  • Gilmartin GM, Nevins JR. An ordered pathway of assembly of components required for polyadenylation site recognition and processing. Genes Dev. 1989 Dec;3(12B):2180–2190. [PubMed]
  • Gilmartin GM, Nevins JR. Molecular analyses of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the pre-mRNA. Mol Cell Biol. 1991 May;11(5):2432–2438. [PMC free article] [PubMed]
  • Gilmartin GM, Fleming ES, Oetjen J, Graveley BR. CPSF recognition of an HIV-1 mRNA 3'-processing enhancer: multiple sequence contacts involved in poly(A) site definition. Genes Dev. 1995 Jan 1;9(1):72–83. [PubMed]
  • Guo Z, Sherman F. 3'-end-forming signals of yeast mRNA. Trends Biochem Sci. 1996 Dec;21(12):477–481. [PubMed]
  • Jenny A, Hauri HP, Keller W. Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit. Mol Cell Biol. 1994 Dec;14(12):8183–8190. [PMC free article] [PubMed]
  • Jenny A, Keller W. Cloning of cDNAs encoding the 160 kDa subunit of the bovine cleavage and polyadenylation specificity factor. Nucleic Acids Res. 1995 Jul 25;23(14):2629–2635. [PMC free article] [PubMed]
  • Jenny A, Minvielle-Sebastia L, Preker PJ, Keller W. Sequence similarity between the 73-kilodalton protein of mammalian CPSF and a subunit of yeast polyadenylation factor I. Science. 1996 Nov 29;274(5292):1514–1517. [PubMed]
  • Keller W. No end yet to messenger RNA 3' processing! Cell. 1995 Jun 16;81(6):829–832. [PubMed]
  • Keller W, Bienroth S, Lang KM, Christofori G. Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3' processing signal AAUAAA. EMBO J. 1991 Dec;10(13):4241–4249. [PubMed]
  • Lutz CS, Alwine JC. Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal. Genes Dev. 1994 Mar 1;8(5):576–586. [PubMed]
  • Lutz CS, Murthy KG, Schek N, O'Connor JP, Manley JL, Alwine JC. Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro. Genes Dev. 1996 Feb 1;10(3):325–337. [PubMed]
  • MacDonald CC, Wilusz J, Shenk T. The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location. Mol Cell Biol. 1994 Oct;14(10):6647–6654. [PMC free article] [PubMed]
  • Manley JL. A complex protein assembly catalyzes polyadenylation of mRNA precursors. Curr Opin Genet Dev. 1995 Apr;5(2):222–228. [PubMed]
  • Manley JL, Takagaki Y. The end of the message--another link between yeast and mammals. Science. 1996 Nov 29;274(5292):1481–1482. [PubMed]
  • McDevitt MA, Hart RP, Wong WW, Nevins JR. Sequences capable of restoring poly(A) site function define two distinct downstream elements. EMBO J. 1986 Nov;5(11):2907–2913. [PubMed]
  • Minvielle-Sebastia L, Winsor B, Bonneaud N, Lacroute F. Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein. Mol Cell Biol. 1991 Jun;11(6):3075–3087. [PMC free article] [PubMed]
  • Minvielle-Sebastia L, Preker PJ, Keller W. RNA14 and RNA15 proteins as components of a yeast pre-mRNA 3'-end processing factor. Science. 1994 Dec 9;266(5191):1702–1705. [PubMed]
  • Murthy KG, Manley JL. Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus. J Biol Chem. 1992 Jul 25;267(21):14804–14811. [PubMed]
  • Murthy KG, Manley JL. The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation. Genes Dev. 1995 Nov 1;9(21):2672–2683. [PubMed]
  • Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. [PubMed]
  • Proudfoot N. Poly(A) signals. Cell. 1991 Feb 22;64(4):671–674. [PubMed]
  • Ryner LC, Takagaki Y, Manley JL. Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly. Mol Cell Biol. 1989 Apr;9(4):1759–1771. [PMC free article] [PubMed]
  • Ryner LC, Takagaki Y, Manley JL. Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3'-end formation. Mol Cell Biol. 1989 Oct;9(10):4229–4238. [PMC free article] [PubMed]
  • Skolnik-David H, Moore CL, Sharp PA. Electrophoretic separation of polyadenylation-specific complexes. Genes Dev. 1987 Sep;1(7):672–682. [PubMed]
  • Smith DB, Davern KM, Board PG, Tiu WU, Garcia EG, Mitchell GF. Mr 26,000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8703–8707. [PubMed]
  • Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. [PubMed]
  • Stumpf G, Domdey H. Dependence of yeast pre-mRNA 3'-end processing on CFT1: a sequence homolog of the mammalian AAUAAA binding factor. Science. 1996 Nov 29;274(5292):1517–1520. [PubMed]
  • Tacke R, Manley JL. The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities. EMBO J. 1995 Jul 17;14(14):3540–3551. [PubMed]
  • Takagaki Y, Ryner LC, Manley JL. Four factors are required for 3'-end cleavage of pre-mRNAs. Genes Dev. 1989 Nov;3(11):1711–1724. [PubMed]
  • Takagaki Y, Manley JL, MacDonald CC, Wilusz J, Shenk T. A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs. Genes Dev. 1990 Dec;4(12A):2112–2120. [PubMed]
  • Takagaki Y, MacDonald CC, Shenk T, Manley JL. The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1403–1407. [PubMed]
  • Takagaki Y, Manley JL. A human polyadenylation factor is a G protein beta-subunit homologue. J Biol Chem. 1992 Nov 25;267(33):23471–23474. [PubMed]
  • Takagaki Y, Manley JL. A polyadenylation factor subunit is the human homologue of the Drosophila suppressor of forked protein. Nature. 1994 Dec 1;372(6505):471–474. [PubMed]
  • Takagaki Y, Seipelt RL, Peterson ML, Manley JL. The polyadenylation factor CstF-64 regulates alternative processing of IgM heavy chain pre-mRNA during B cell differentiation. Cell. 1996 Nov 29;87(5):941–952. [PubMed]
  • Tsai DE, Harper DS, Keene JD. U1-snRNP-A protein selects a ten nucleotide consensus sequence from a degenerate RNA pool presented in various structural contexts. Nucleic Acids Res. 1991 Sep 25;19(18):4931–4936. [PMC free article] [PubMed]
  • Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. [PubMed]
  • Wahle E, Keller W. The biochemistry of polyadenylation. Trends Biochem Sci. 1996 Jul;21(7):247–250. [PubMed]
  • Wilusz J, Shenk T. A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif. Cell. 1988 Jan 29;52(2):221–228. [PubMed]
  • Wilusz J, Shenk T, Takagaki Y, Manley JL. A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates. Mol Cell Biol. 1990 Mar;10(3):1244–1248. [PMC free article] [PubMed]
  • Wilusz J, Shenk T. A uridylate tract mediates efficient heterogeneous nuclear ribonucleoprotein C protein-RNA cross-linking and functionally substitutes for the downstream element of the polyadenylation signal. Mol Cell Biol. 1990 Dec;10(12):6397–6407. [PMC free article] [PubMed]

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