Gary Brewer

AUF1 binding affinity to A+U-rich elements correlates with rapid mRNA degradation.

Rapid degradation of many labile mRNAs is regulated in part by an A+U-rich element (ARE) in their 3′-untranslated regions. Extensive mutational analyses of various AREs have identified important components of the ARE, such as the nonamer motif UUAUUUAUU, two copies of which serve as a potent mRNA destabilizer. To investigate the roles of trans-acting factors in ARE-directed mRNA degradation, we previously purified and molecularly cloned the RNA-binding protein AUF1 and demonstrated that both cellular and recombinant AUF1 bind specifically to AREs as shown by UV cross-linking assays in vitro. In the present work, we have examined the in vitro RNA-binding properties of AUF1 using gel mobility shift assays with purified recombinant His6-AUF1 fusion protein. We find that ARE binding affinities of AUF1 correlate with the potency of an ARE to direct degradation of a heterologous mRNA. These results support a role for AUF1 in ARE-directed mRNA decay that is based upon its affinity for different AREs.

Assembly of AUF1 oligomers on U-rich RNA targets by sequential dimer association.

Many labile mammalian mRNAs are targeted for rapid cytoplasmic turnover by the presence of A + U-rich elements (AREs) within their 3′-untranslated regions. These elements are selectively recognized by AUF1, a component of a multisubunit complex that may participate in the initiation of mRNA decay. In this study, we have investigated the recognition of AREs by AUF1 in vitro using oligoribonucleotide substrates. Gel mobility shift assays demonstrated that U-rich RNA targets were specifically bound by AUF1, generating two distinct RNA-protein complexes in a concentration-dependent manner. Chemical cross-linking revealed the interaction of AUF1 dimers to form tetrameric structures involving protein-protein interactions in the presence of high affinity RNA targets. From these data, a model of AUF1 association with AREs involving sequential dimer binding was developed. Using fluorescent RNA substrates, binding parameters of AUF1 dimer-ARE and tetramer-ARE equilibria were evaluated in solution by fluorescence anisotropy measurements. Using two AUF1 deletion mutants, sequences C-terminal to the RNA recognition motifs are shown to contribute to the formation of the AUF1 tetramer·ARE complex but are not obligate for RNA binding activity. Kinetic studies demonstrated rapid turnover of AUF1·ARE complexes in solution, suggesting that these interactions are very dynamic in character. Taken together, these data support a model where ARE-dependent oligomerization of AUF1 may function to nucleate the formation of a trans-acting, RNA-destabilizing complex in vivo.

Structural Determinants in AUF1 Required for High Affinity Binding to A + U-rich Elements

AUF1 is an RNA-binding protein that contains two nonidentical RNA recognition motifs (RRMs). AUF1 binds to A + U-rich elements (AREs) with high affinity. The binding of AUF1 to AREs is believed to serve as a signal to an mRNA-processing pathway that degrades mRNAs encoding many cytokines, oncoproteins, and G protein-coupled receptors. Because the ARE binding activity of AUF1 appears central to the regulation of many important genes, we analyzed the domains of the protein that are important for this activity. Examination of the RNA binding affinity of various AUF1 mutants suggests that both RRMs may be required for binding to the human c-fos ARE. However, the two RRMs together are not sufficient. Highest affinity binding of AUF1 to an ARE requires an alanine-rich region of the N terminus and a short glutamine-rich region in the C terminus. In addition, the N terminus is required for dimerization of AUF1. However, AUF1 binds an ARE as a hexameric protein. Thus, protein-protein interactions are important for high affinity ARE binding activity of AUF1.

Developmental Regulation of RNA Transcript Destabilization by A + U-rich Elements is AUF1-dependent

The developmental immaturity of neonatal phagocytic function is associated with decreased accumulation and half-life (t 1 2 ) of granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA in mononuclear cells (MNC) from the neonatal umbilical cord compared with adult peripheral blood. The in vivo t 1 2 of GM-CSF mRNA is 3-fold shorter in neonatal (30 min) than in adult (100 min) MNC. Turnover of mRNA containing a 3′-untranslated region (3′-UTR) A + U-rich element (ARE), which regulates GM-CSF mRNA stability, is accelerated in vitro by protein fractions enriched for AUF1, an ARE-specific binding factor. The data reported here demonstrate that the ARE significantly accelerates in vitrodecay of the GM-CSF 3′-UTR in the presence of either neonatal or adult MNC protein. Decay intermediates of the GM-CSF 3′-UTR are generated that are truncated at either end of the ARE. Furthermore, thet 1 2 of the ARE-containing 3′-UTR is 4-fold shorter in the presence of neonatal (19 min) than adult (79 min) MNC protein, reconstituting developmental regulation in a cell-free system. Finally, accelerated ARE-dependent decay of the GM-CSF 3′-UTRin vitro by neonatal MNC protein is significantly attenuated by immunodepletion of AUF1, providing new evidence that this accelerated turnover is ARE- and AUF1-dependent.

Regulation of AUF1 Expression via Conserved Alternatively Spliced Elements in the 3′ Untranslated Region

ABSTRACT The A+U-rich RNA-binding factor AUF1 exhibits characteristics of atrans-acting factor contributing to the rapid turnover of many cellular mRNAs. Structural mapping of the AUF1 gene and its transcribed mRNA has revealed alternative splicing events within the 3′ untranslated region (3′-UTR). In K562 erythroleukemia cells, we have identified four alternatively spliced AUF1 3′-UTR variants, including a population of AUF1 mRNA containing a highly conserved 107-nucleotide (nt) 3′-UTR exon (exon 9) and the adjacent downstream intron (intron 9). Functional analyses using luciferase–AUF1 3′-UTR chimeric transcripts demonstrated that the presence of either a spliceable or an unspliceable intron 9 in the 3′-UTR repressed luciferase expression incis, indicating that intron 9 sequences may down-regulate gene expression by two distinct mechanisms. In the case of the unspliceable intron, repression of luciferase expression likely involved two AUF1-binding sequences, since luciferase expression was increased by deletion of these sites. However, inclusion of the spliceable intron in the luciferase 3′-UTR down-regulated expression independent of the AUF1-binding sequences. This is likely due to nonsense-mediated mRNA decay (NMD) owing to the generation of exon-exon junctions more than 50 nt downstream of the luciferase termination codon. AUF1 mRNA splice variants generated by selective excision of intron 9 are thus also likely to be subject to NMD since intron 9 is always positioned >137 nt downstream of the stop codon. The distribution of alternatively spliced AUF1 transcripts in K562 cells is consistent with this model of regulated AUF1 expression.

Characterization of cDNAs encoding the murine A+U-rich RNA-binding protein AUF1.

A+U-rich elements (ARE) serve to control the degradation of some proto-oncogene and lymphokine mRNAs. The protein, AUF1, which consists of two polypeptides of 37 and 40 kDa (p37 and p40, respectively) when purified from cytosol, has been implicated in ARE-directed mRNA turnover due to its binding to ARE. Molecular cloning of a cDNA (p37AUF1) corresponding to human p37 predicted a polypeptide containing two non-identical RNA recognition motifs (RRM) and a C-terminal Gln-rich domain [Zhang et al. Mol. Cell. Biol. 13 (1993) 7652-7665]. Two cDNAs, designated muAUF1-3 and muAUF1-7, were isolated from a murine fetal cDNA library, using as a probe, a fragment of the p37AUF1 cDNA encoding RRM1 and approximately half of RRM2. The muAUF1-3 open reading frame (ORF) was very homologous to human p37AUF1 with the greatest homology between the corresponding RRMs and the C-terminal Gln-rich motif. Clone muAUF1-7 was highly homologous to muAUF1-3, but was truncated within the region encoding the RNP-1 box in RRM2. Clone muAUF1-3 encoded 19 amino acids in RRM1 not encoded by either muAUF1-7 or human p37AUF1. Such alterations in sequence could modify the RNA-binding properties of these proteins and have concomitant effects on ARE-directed posttranscriptional processes.

Evidence for a 3′-5′ Decay Pathway for c-myc mRNA in Mammalian Cells

Many mRNAs in mammalian cells decay via a sequential pathway involving rapid conversion of polyadenylated molecules to a poly(A)-deficient state followed by rapid degradation of the poly(A)-deficient molecules. However, the rapidity of this latter step(s) has precluded further analyses of the decay pathways involved. Decay intermediates derived from degradation of poly(A)-deficient molecules could offer clues regarding decay pathways, but these intermediates have not been readily detected. Cell-free mRNA decay systems have proven useful in analyses of decay pathways because decay intermediates are rather stable in vitro. Cell-free systems indicate that many mRNAs decay by a sequential 3′-5′ pathway because 3′-terminal decay intermediates form following deadenylation. However, if 3′-terminal, in vitro decay intermediates reflect a biologically significant aspect of mRNA turnover, then similar intermediates should be present in cells. Here, I have compared the in vivo and in vitro decay of mRNA encoded by the c-myc proto-oncogene. Its decay bothin vivo and in vitro occurs by rapid removal of the poly(A) tract and generation of a 3′-terminal decay intermediate. These data strongly suggest that a 3′-5′ pathway contributes to turnover of c-myc mRNA in cells. It is likely that 3′-5′ decay represents a major turnover pathway in mammalian cells.

MOLECULAR ANALYSIS OF DECREASED GM-CSF mRNA STABILITY IN NEONATAL VS. ADULT MONONUCLEAR CELLS: ASSOCIATION WITH INCREASED LEVELS OF SPECIFIC AUUUA-BINDING AUF1 PROTEINS AND INCREASED IN VITRO DEGRADATION.

Expression of GM-CSF mRNA is 4-fold lower in activated peripheral blood mononuclear cells (MNC) from newborn (Nb) compared to adult (Ad) (Cairo, et al, Pediatr Res, 30:362, 1991). While the GM-CSF transcription rate is similar in Nb and Ad MNC, transcript half-life is 3-fold less in Nb activated MNC. Inhibiting protein synthesis with 10μg/mL cycloheximide(CHX; 3h) after stimulation with 20ng/mL PMA + ≈2μg/mL PHA (3h) increased GM-CSF mRNA 3-fold in Nb MNC but had only a minimal effect in Ad MNC compared to PMA+PHA. To investigate the involvement of RNA binding proteins, such as the cloned AUF1 factor (Zhang, et al., Mol Cell Biol, 13:7652, 1993) interacting with 8 AUUUA-motifs (AU) in the 3′-untranslated region (8AU), cytoplasmic extracts were prepared from Nb vs. Ad MNC unstimulated or stimulated with PMA+PHA (6h), ±CHX (3h). Electrophoretic mobility shift assays using 32P-labeled 8AU RNA revealed two, RNaseT1-resistant, bound complexes from both Nb and Ad MNC extracts, and competitive assays localized the binding site of both complexes to the region containing 7 of 8 AU-motifs. One of the bound complexes was twice as abundant in Nb than in Ad MNC. Inclusion of AUF1 antiserum revealed a super-shifted complex at 30-fold higher levels in Nb than Ad MNC extracts. A human carcinoma cell line (5637) with extended GM-CSF mRNA half-life also showed very low levels of anti-AUF1 super-shifted complex. Anti-AUF1 immunoblotting showed considerably higher levels of the two smallest of four AUF1 protein species in Nb than in Ad MNC or 5637 extracts. Additionally,in vitro mRNA decay assays found the stability of 32P-8AU to be 85% less in Nb than Ad MNC extracts compared to transcripts lacking AU. These results suggest that destabilization of GM-CSF mRNA in Nb MNC is translation-dependent and that increased levels of specific AU-binding AUF1 species in Nb MNC may target transcripts for increased degradation which could account, in part, for dysregulation of Nb phagocytic immunity.