The ARE-associated factor AUF1 binds poly(A) in vitro in competition with PABP.
The ARE (AU-rich element) is a post-transcriptional element controlling both mRNA turnover and translation initiation by primarily inducing poly(A) tail shortening. The mechanisms by which the ARE-associated proteins induce deadenylation are still obscure. One possibility among others would be that an ARE-ARE-BP (ARE-binding protein) complex intervenes in the PABP [poly(A)-binding protein]-poly(A) tail association and facilitates poly(A) tail accessibility to deadenylases. Here, we show by several experimental approaches that AUF1 (AU-rich element RNA-binding protein 1)/hnRNP (heterogeneous nuclear ribonucleoprotein) D, an mRNA-destabilizing ARE-BP, can bind poly(A) sequence in vitro. First, endogenous AUF1 proteins from HeLa cells specifically bound poly(A), independently of PABP. Secondly, using polyadenylated RNA probes, we showed that (i) the four recombinant AUF1 isoforms bind poly(A) as efficiently as PABP, (ii) the AUF1 binding to poly(A) does not change when the polyadenylated probe contains the GM-CSF (granulocyte/macrophage-colony stimulating factor) ARE, suggesting that, in vitro, the AUF1-poly(A) association was independent of the ARE sequence itself. In vitro, the binding of AUF1 isoforms to poly(A) displayed oligomeric and co-operative properties and AUF1 efficiently displaced PABP from the poly(A). Finally, the AUF1 molar concentration in HeLa cytoplasm was only 2-fold lower than that of PABP, whereas in the nucleus, its molar concentration was similar to that of PABP. These in vitro results suggest that, in vivo, AUF1 could compete with PABP for the binding to poly(A). Altogether, our results may suggest a role for AUF1 in controlling PABP-poly(A) tail association.
Sagliocco
Francis
F
INSERM, E362, Bordeaux, F-33076 France.
Laloo
Benoît
B
Cosson
Bertrand
B
Laborde
Laurence
L
Castroviejo
Michel
M
Rosenbaum
Jean
J
Ripoche
Jean
J
Grosset
Christophe
C
D001667:Binding, Competitive; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011061:Poly A / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
C000630986:HNRNPD protein, human; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D039102:Poly(A)-Binding Proteins; D011994:Recombinant Proteins; D011061:Poly A