Novel Binding of HuR and Poly(C)-binding Protein to a Conserved UC-rich Motif within the 3′-Untranslated Region of the Androgen Receptor Messenger RNA

Abstract

The interaction between androgens such as dihydrotestosterone (DHT) 1 and the androgen receptor (AR) plays a crucial role in the evolution of human prostate cancer, and modulation of this interaction represents a major therapeutic strategy (1, 2). The biological action of androgen is mediated via binding to the AR, a member of the nuclear hormone receptor family of transcription factors that regulate expression of androgen-responsive genes (3). Androgens promote the growth of prostate cancer cells, and androgen deprivation results in regression of most prostate cancers, although this effect is eventually abrogated by the emergence of androgen-insensitive disease. In contrast, AR expression correlates with better clinical outcome in human breast cancer (4, 5), and androgens have been shown to inhibit the growth of breast cancer cells expressing the AR (6, 7). To better understand the mechanisms underlying the expression of the AR gene, we recently utilized LNCaP prostate cancer cells and MDA453 breast cancer cells to determine the relative importance of transcriptional and post-transcriptional mechanisms in androgen-regulated AR gene expression (8). In LNCaP cells, androgens down-regulate transcription of the AR gene, but stabilize AR mRNA. In MDA453 cells, androgens produce rapid destabilization of AR mRNA in the absence of transcriptional change. These data suggest an important role for post-transcriptional events (in particular, AR mRNA turnover) in the regulation of AR gene expression in prostate and breast cancer cells. We therefore decided to investigate the mechanisms regulating AR mRNA stability. mRNA turnover is a central mechanism in the control of gene expression (9), where interactions between specific sequences within mRNA (cis-acting elements) and cellular RNA-binding proteins (trans-acting factors) regulate ribonuclease action and subsequent mRNA decay rates. Several cis-acting elements have been described, and the best characterized is the adenylate/uridylate (AU)-rich element (ARE). The ARE most often resides in the 3Ј-untranslated region (3Ј-UTR) and contains AUUUA pentamers and/or UUAUUUA(U/A)(U/A) nonamers

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

“…12 The NMR titration data herein reported and earlier findings with HuB, 59 demonstrate that isolated RRM3 can bind to AREs, although previous studies suggested a negligible role of HuR RRM3 ¡ as part of HuR FL ¡ in AREs recognition. 34,[44][45][46] Isolated RRM3 is in a monomer/dimer exchange on a time scale unfavorable for NMR observation of the residues at the dimerization site, notably those in the W261-T271 region at the C-end of helix a 1 . Indeed, this region shows large fluctuations in MD simulations, which affect the H-bonding pattern at a 1 and the dynamics of W261 ring.…”

“…Interestingly, isolated HuR RRM3 preferably binds U-rich versus AU-rich RNA stretches, despite previous studies suggesting that RRM3 has a negligible contribution to ARE-binding events in comparison with the other 2 HuR RRM motifs. 34,[44][45][46] Although the HuR linker between RRM2 and RRM3 could contribute to the ARE stabilization, 34,35 it was unfeasible to obtain a linker-bearing RRM3 construct in a soluble manner. Moreover, HuR RRM3 phosphorylation, which was mimicked by a S318-to-D318 substitution, causes no significant structural changes of RRM3, in contrast to a phosphorylation-mediated unfolding described in other types of RNA domains.…”

“…43 Cytoplasmic binding of HuR to single-stranded ARE-containing mRNAs is mainly driven by RRM1 and RRM2 domains. 34,[44][45][46] RRM3 is involved in binding long poly-A tails of mRNAs [47][48][49] and in catalyzing the 3 0 -terminal adenosyl modification of non-polyadenylated RNA substrates. 50 Besides its role in RNA recognition, RRM3 is also responsible for the formation of HuR multimers, 34 as occurs with the homologous Drosophila ELAV protein.…”

The Acute Box cis-Element in Human Heavy Ferritin mRNA 5′-Untranslated Region Is a Unique Translation Enhancer That Binds Poly(C)-binding Proteins

“…GST Fusion Protein Preparation-Purified recombinant ␣CP1 from PreScission protease-cleaved GST-␣CP1 fusion protein was prepared as described previously (18). Briefly, 1-liter cultures of Escherichia coli DH5␣ expressing GST or GST-␣CP1 fusion constructs (described in Ref.…”

“…The poly(C)-binding proteins (PCBPs) are a structurally diverse family, including heterogeneous nuclear ribonucleoprotein K and ␣CP1-4 (17). They bind mRNA sequences that contain either a single C run (heterogeneous nuclear ribonucleoprotein K) or stretch of C's (␣CPs) via their K-homology (KH) domains (18). The PCBPs have been implicated in regulation of mRNA stability, translation silencing (mainly through interactions with 3Ј-UTR sequences), and enhancement.…”