Principles of HuR-RNA targeting, interaction dynamics, and functional outcomes

In recent years, the pervasiveness and importance of post-transcriptional regulation has reshaped the underlying principles of the organizational logic of gene expression. RNA-binding proteins (RBPs) and non-coding RNAs are the regulatory molecules primary responsible for interaction with target mRNAs and thereby regulating post-transcriptional processes eventually influencing characteristics of the encoded protein. Many of the mRNA targets of RBPs encode functionally related proteins, which for post-trascriptional operons, resulting in coordination of macromolecular complexes or specific cellular processes. Thus, identifying RNA targets, precise binding sites, and the dynamics of these interactions will reveal how these important regulatory factors contribute to gene regulatory networks.

ELAV family of human RBPs consist of 4 members, which all have 3 RRM (RNA-recognition motif) domains the last separated by a hinge region. It predominant role is to positively regulate the stability and translation of target mRNAs through binding to ARE (AU-rich elements) in the 3' UTR (untranslated region) of protein coding transcripts. In response to certain stimuli, HuR is subject to post-translational modifications and changes subcellular localization, which impacts its regulatory capacity. In this study on a transcriptome-wide level, we interrogate the RNA targets, precise binding sites, as well as the remodeling of these interactions in response to stimuli.

We utilized two complementary methods, RIP-chip and PAR-CLIP, to identify targets of HuR and high-resolution binding sites on a transcriptome-wide scale. We discovered that HuR-mRNA interactions are not restricted to the 3' UTR and there are thousands of intronic binding sites. A significant proportion of intronic binding sites are contained in the poly-pyrimidine tract near 3' splice sites. Binding sites in the 3' UTR and intron are often approximately 30 nucleotides apart. HuR can bind to both AU-rich and U-rich sequences, the former more prevalent in 3' UTRs and the latter more prevalent at the 3' splice site.

Next we integrated the binding data with transcriptomics of HuR siRNA mediated knockdown. We found that the degree of binding is proportional to the degree of HuR-dependent stabilization. Moreover the ability to stabilize mRNA is not restricted to 3' UTR binding sites, as intronic binding sites also exhibited the binding degree correlated stabilization. We observed that the spatial pattern of HuR binding sites relative to exons influences exon usage decisions. Specifically, binding sites upstream of the exon promote exclusion, while binding sites downstream of the exon promote inclusion.