Identification of a target RNA motif for RNA-binding protein HuR

Abstract

HuR, a protein that binds to specific mRNA subsets, is increasingly recognized as a pivotal posttranscriptional regulator of gene expression. Here, HuR was immunoprecipitated under conditions that preserved HuR-RNA interactions, and HuR-bound target mRNAs were identified by cDNA array hybridization. Analysis of primary sequences and secondary structures shared among HuR targets led to the identification of a 17- to 20-base-long RNA motif rich in uracils. This HuR motif was found in almost all mRNAs previously reported to be HuR targets, was located preferentially within 3' untranslated regions of all unigene transcripts examined, and was conserved in >50% of human and mouse homologous genes. Importantly, the HuR motif allowed the successful prediction and subsequent validation of novel HuR targets from gene databases. This study describes an HuR target RNA motif and presents a general strategy for identifying target motifs for RNA-binding proteins.

Fig. 1.

Identification of HuR target mRNAs by IP of HuR-containing mRNP complexes and cDNA array hybridization with probes derived from RNA in the IP material. IP assay using 3 mg of protein lysate prepared from ActD-treated RKO cells and 30 μg of either anti-HuR antibody or IgG1, under conditions that preserved mRNP complexes. (A) HuR was detected by Western blotting in aliquots of IP material. H.C., heavy chain; L.C., light chain. (B) Representative images obtained after reverse-transcribing mRNAs in the IP material and hybridizing it to human cDNA arrays. Solid arrowheads, signals enriched in samples obtained in the HuR IP; open arrowheads, nonspecific signals, present in both IP materials (IgG1 and HuR).

Fig. 2.

Sequence and structure of the predicted HuR motif. (A) Probability matrix (graphic logo) depicting the relative frequency of finding each residue at each position within the motif, elucidated from the array-derived experimental data set. (B) Structural alignment of eight examples of the HuR motif in specific mRNAs; the corresponding gene name and motif score are shown. (C) Secondary structure of three representative examples of the HuR motif.

Fig. 3.

Validation of novel HuR targets predicted by using the HuR motif. (A) Biotin pull-down assays to assess the ability of endogenous HuR to bind to biotinylated transcripts of interest. The indicated biotinylated transcripts were incubated with cytoplasmic protein lysate from ActD-treated RKO cells, whereupon their association with HuR was detected by Western blotting; stripping and rehybridization of blots to detect β-tubulin (β-Tub) revealed an absence of contaminating proteins in the pull-down material. (B) Three biotinylated transcripts (heavy bars) spanning different regions of HLF mRNA, which bears nine hits for the HuR motif (solid circles) with varying scores (parentheses), were used in pull-down assays to detect their association with HuR as described in A. (C) The binding of endogenous HuR with endogenous target mRNAs was further tested in RKO cells by IP, followed by detection of the target transcripts of interest by low-cycle (25-30) RT-PCR amplification of IP material. PCR products were visualized by electrophoresis in ethidium bromide-stained 1% agarose gels. (D) The abundance of transcripts present in material obtained from HeLa cells after either HuR IP or IgG1 IP was assessed by real-time RT-PCR, and fold differences were plotted on a log scale.