Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1.

Abstract

The degradation of some proto-oncogene and lymphokine mRNAs is controlled in part by an AU-rich element (ARE) in the 3' untranslated region. It was shown previously (G. Brewer, Mol. Cell. Biol. 11:2460-2466) that two polypeptides (37 and 40 kDa) copurified with fractions of a 130,000 x g postribosomal supernatant (S130) from K562 cells that selectively accelerated degradation of c-myc mRNA in a cell-free decay system. These polypeptides bound specifically to the c-myc and granulocyte-macrophage colony-stimulating factor 3' UTRs, suggesting they are in part responsible for selective mRNA degradation. In the present work, we have purified the RNA-binding component of this mRNA degradation activity, which we refer to as AUFl. Using antisera specific for these polypeptides, we demonstrate that the 37-and 40-kDa polypeptides are immunologically cross-reactive and that both polypeptides are phosphorylated and can be found in a complex(s) with other polypeptides. Immunologically related polypeptides are found in both the nucleus and the cytoplasm. The antibodies were also used to clone a cDNA for the 37-kDa polypeptide. This cDNA contains an open reading frame predicted to produce a protein with several features, including two RNA recognition motifs and domains that potentially mediate protein-protein interactions. These results provide further support for a role of this protein in mediating ARE-directed mRNA degradation.The c-myc gene is important for the control of cellular growth, differentiation, and transformation (reviewed in references 17 and 41). It belongs to the class of immediateearly genes whose expression is required to drive cells from Go to G1 following stimulation of quiescent cells by growth factors. However, the c-myc gene is not unique in terms of having an essential role in cellular growth processes. It has been known for decades that specific and timely changes in the expression of multiple genes are required for proper embryonic development and cell maturation (20). Expression of genes such as c-myc seems to be regulated not only at the levels of transcription, attenuation, nuclear processing, and translation but also at the level of mRNA turnover (reviewed in reference 41). Indeed, direct half-life measurements indicated that c-myc mRNA has a half-life of 15 to 40 min (19). These and other studies (41) demonstrated that the control of c-myc mRNA turnover might be an important means of regulating both the level and the timing of c-myc expression.Many proto-oncogene mRNAs are very unstable. The rapid turnover of c-myc mRNA is controlled by sequences in the 3' untranslated region (3'UTR) or by coding region sequences (reviewed in references 33 and 60). A common feature of many labile mRNAs, such as those for c-myc, c-fos, and granulocyte-macrophage colony-stimulating factor (GM-CSF), is the presence of an AU-rich element (ARE) in the 3'UTR which is one cis-acting element responsible for their rapid degradation (reviewed in reference 3, 48, and 60). It AUUUA (70). This might be important because...

Developmental expression of AUF1 and HuR, two c-myc mRNA binding proteins

“…(a) silver staining. (b) ECL detection after incubation with anti-AUF1 antibodies AUF1, HuR and c-myc mRNA expression in vivo I Lafon et al 1995; Zhang et al, 1993) revealed two in frame insertions, one 57 bp long in the 5' coding region and another, 147 bp long, in the 3' coding region ( Figure 3A). These variations, which result from alternative pre-mRNA splicing (Wagner et al, 1998), lead either to a 19 amino-acid (19-aa) insertion in the N-terminal portion of RRM1 or to a 49-amino acid (49-aa) insertion in the C-terminal region of the AUF1 isoforms, respectively (see Figure 3A).…”

“…The AUF1 antibodies used in the Western blot assay initially revealed three immunologically related polypeptides of 37 (p37), 40 (p40) and 45 (p45) kDa in extracts from the human K562 cell line (Zhang et al, 1993). These three polypeptides are also observed in murine tissues, though their relative abundance is not identical.…”

“…Although several polypeptides reacting either with anti-AUF1 (Zhang et al, 1993) or anti-hnRNP antibodies (Ishikawa et al, 1993) were previously described, their exact nature is not fully understood. They could either be encoded by di erent mRNAs or correspond to di erent posttranslational modi®cations of the same polypeptide.…”

“…AUF1 (Brewer, 1991;Zhang et al, 1993), also called hnRNP D0 (Kajita et al, 1995), was identi®ed due to its ability to induce c-myc mRNA destabilization in vitro (Brewer, 1991). It consists of two polypeptides of 37 and 40 kDa containing two RRMs (RNA Recognition Motifs) and a glutamine-rich motif present in their carboxy-terminal regions which might mediate proteinprotein interactions (Ehrenman et al, 1994;Zhang et al, 1993) and that is important for ARE-binding .…”

“…It consists of two polypeptides of 37 and 40 kDa containing two RRMs (RNA Recognition Motifs) and a glutamine-rich motif present in their carboxy-terminal regions which might mediate proteinprotein interactions (Ehrenman et al, 1994;Zhang et al, 1993) and that is important for ARE-binding . UV cross-linking assays have shown that AUF1 binds the AREs present in the c-fos and c-myc proto-oncogene mRNAs and in the granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine mRNA.…”