Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM

Abstract

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate–rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3′ untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.

RNA-Binding Proteins as Important Regulators of Long Non-Coding RNAs in Cancer

“…The human antigen R (HuR), the protein product of the ELAV1 gene, is a ubiquitously expressed RBP that contains three RNA recognition motifs (RRMs) via which it preferentially binds to adenylate/uridylate-rich RNA elements (AREs) [71][72][73]. AREs are signals for rapid RNA degradation, and by blocking these recognition sites HuR can stabilize its RNA interaction partners [71][72][73].…”

“…The human antigen R (HuR), the protein product of the ELAV1 gene, is a ubiquitously expressed RBP that contains three RNA recognition motifs (RRMs) via which it preferentially binds to adenylate/uridylate-rich RNA elements (AREs) [71][72][73]. AREs are signals for rapid RNA degradation, and by blocking these recognition sites HuR can stabilize its RNA interaction partners [71][72][73]. HuR is frequently upregulated in cancer cells and is known to be involved in many hallmarks of cancer, such as invasion, angiogenesis, and inflammation, by post-transcriptionally regulating various cancer-related mRNAs [71,72,[74][75][76].…”

“…All four AUF1 isoforms form dimers and contain two tandem RRMs that include RNP sequence motifs [91]. Via these domains AUF1 primarily, but not exclusively, binds to U-rich RNA sequences of AREs, therefore competing with HuR [61,[71][72][73]85,91]. In addition, it has been found to bind to non-canonical U-rich and GU-rich sites [61,91].…”

“…Besides the two previously discussed examples HuR and AUF1, TTP is another RBP that binds to AREs, adenylate/uridylate-rich RNA motifs that function as signals for the rapid degradation of RNA [61,66,[71][72][73]85,91]. While HuR promotes the stability of RNAs by shielding these recognition sites, AUF1 recruits, upon binding to AREs, components of the RNA degradation machinery [71][72][73]91]. The exact components are not yet known [91].…”

The mRNA-binding protein HuR is a kinetically-privileged electrophile sensor

“…HuR is a multidomain protein harboring a total of 3 cysteines (Figure 2A), one of which (C13) resides close to the unstructured N-terminus, [30] and two of which (C245 and C284), also appear to be solvent accessible, at least based on crystal and NMR solution structures of the RRM3 domain wherein these two cysteines reside ( Figure 2B). [31][32][33] Cysteine 13 (C13) of human HuR has been previously proposed to act as a redox sensor that functions through formation of a disulfide bridge between two molecules of HuR. This postulate was based on gel filtration analysis of a truncated HuR construct (Residues 2-189, spanning the unstructured N-terminal region housing C13 and the first two RNArecognition motif [RRM] domains; Figure 2A).…”