AUF1 Binding Affinity to A+U-rich Elements Correlates with Rapid mRNA Degradation

Abstract

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 His 6 -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.Control of mRNA stability is an important component of eukaryotic gene expression and involves cis-acting elements that can be found in the coding region and/or UTRs 1 of mRNAs (reviewed in Refs. 1-6). One type of cis-acting instability element is comprised of the AREs found in the 3Ј-UTRs of many unstable mRNAs (reviewed in Ref. 7). Many ARE-containing mRNAs are degraded by a sequential pathway involving removal of the poly(A) tract followed by degradation of the mRNA body (8 -10). In most cases the poly(A) tract is thought to protect the mRNA from ribonuclease attack so that its removal permits degradation of the mRNA body (reviewed in Ref. 11). While it has been known for almost a decade that AREs are important for mRNA instability (12-15), the mechanism(s) by which they mediate mRNA turnover is still unknown.Despite the presence of AREs in many different mRNAs, there is no single evolutionarily conserved AϩU-rich instability sequence. Typically AREs contain multiple copies of the pentanucleotide AUUUA, often in conjunction with one or more U-rich regions (14). In addition, transfection studies indicate that as the number of tandemly repeated AUUU motifs is increased in a reporter mRNA, its instability increases. Likewise, two copies of the nonameric motif UUAUUUAUU act as a more potent destabilizer than a single nonameric motif (16,17). Together, these analyses suggest that potent destabilizing AREs are high affinity binding sites for a mRNA decay factor(s).In order to investigate how AREs function in mRNA turnover, we utilized a cell-free mRNA decay system to identify proteins that may be relevant to ARE-directed mRNA decay (8,18,19). To this end, we previously reported the purification, molecular cloning, and characterization of the ARE-binding protein AUF1 (20). Cellular AUF1 purified from cytoplasmic extracts of K562 human erythroid leukemia cells consists of a 37-and a 40-kDa isoform. Cloning of the 37-kDa isoform, p37 AUF1 , revealed two nonidentical RNA recognition motifs (21) and a short glutamine-rich region i...

HuR binding to cytoplasmic mRNA is perturbed by heat shock

“…3 and 4) is consistent with the possibility that once bound, it targets an mRNA for such rapid decay that it is immediately released (26). Although there have been many reports of hnRNP D being UV crosslinked to ARE sequences in vitro (42), our negative in vivo results suggest that caution should be applied to the interpretation of these observations.…”

“…3, hnRNP blot, A1 37°C, fractions 15-17). Surprisingly, despite its cytoplasmic abundance and its ability to crosslink to ARE sequences in vitro (42), an even tinier fraction of hnRNP D (about 0.05% above background) cosediments with polysomes ( Fig. 3, hnRNP D blot, 37°C, fractions 15-16).…”

MAPK regulation of maternal and zygotic Notch transcript stability in early development

“…Thus AREs can exert either a stabilizing or destabilizing effect on the mRNA molecule (17,18), depending on the p38MAPK activity within the cell. The strength of the effect varies with the number and configuration of AREs in the transcript (19).…”

Identification of RNA-binding Proteins in RAW 264.7 Cells That Recognize a Lipopolysaccharide-responsive Element in the 3-Untranslated Region of the Murine Cyclooxygenase-2 mRNA

“…Binding of other proteins, such as TIA-1, TIAR, and tristetraprolin, promotes decreased message expression (14 -18). Still other proteins like AUF1 (also known as hnRNP D) may play a role in both degradation and stabilization of target messages (7,19). Adding to the complexity of these interactions is the fact that many of these proteins exist as multiple isoforms that arise because of alternative splicing events, all of which may be subject to post-translational modifications.…”