Comparative 3'UTR Analysis Allows Identification of Regulatory Clusters that Drive Eph/ephrin Expression in Cancer Cell Lines

Jennifer Winter,Albrecht Otto,Sven Krause,Susann Schweiger,Stefan Roepcke,Martin Vingron,Eva-Christina Müller,Thomas Preiss

doi:10.1371/journal.pone.0002780

Abstract

Eph receptors are the largest family of receptor tyrosine kinases. Together with their ligands, the ephrins, they fulfill multiple biological functions. Aberrant expression of Ephs/ephrins leading to increased Eph receptor to ephrin ligand ratios is a critical factor in tumorigenesis, indicating that tight regulation of Eph and ephrin expression is essential for normal cell behavior. The 3'-untranslated regions (3'UTRs) of transcripts play an important yet widely underappreciated role in the control of protein expression. Based on the assumption that paralogues of large gene families might exhibit a conserved organization of regulatory elements in their 3'UTRs we applied a novel bioinformatics/molecular biology approach to the 3'UTR sequences of Eph/ephrin transcripts. We identified clusters of motifs consisting of cytoplasmic polyadenylation elements (CPEs), AU-rich elements (AREs) and HuR binding sites. These clusters bind multiple RNA-stabilizing and destabilizing factors, including HuR. Surprisingly, despite its widely accepted role as an mRNA-stabilizing protein, we further show that binding of HuR to these clusters actually destabilizes Eph/ephrin transcripts in tumor cell lines. Consequently, knockdown of HuR greatly modulates expression of multiple Ephs/ephrins at both the mRNA and protein levels. Together our studies suggest that overexpression of HuR as found in many progressive tumors could be causative for disarranged Eph receptor to ephrin ligand ratios leading to a higher degree of tissue invasiveness.

Highlights

The 3’untranslated regions (3’UTRs) of mRNAs play crucial roles in posttranscriptional regulation of gene expression, for example by modulating mRNA localization [1,2,3], stability [4], and translation [5,6]
Whereas coding regions and exon/intron boundaries [19] are highly conserved between the different members of the Eph receptor and ephrin ligand gene families, the 3’UTRs of transcripts show a high diversity of length and sequence: those of the ephrin ligands vary from 552 bp (EfnA4) to 3171 bp (EfnB2) and those of the Eph receptors from 198 bp (EphB6) to 3310 bp (EphA4) (Fig. 1, Table S1)
We show that several of the Eph/ephrin 3’UTRs, exhibiting substantial divergence between paralogs in terms of length and sequence, contain evolutionarily conserved motif clusters composed of overlapping cytoplasmic polyadenylation elements (CPEs), AU-rich elements (AREs), and HuR binding sites

Summary

Introduction

The 3’untranslated regions (3’UTRs) of mRNAs play crucial roles in posttranscriptional regulation of gene expression, for example by modulating mRNA localization [1,2,3], stability [4], and translation [5,6]. Apart from having binding sites for the recently discovered microRNAs, 3’UTRs can harbor motifs that interact with specific RNA-binding proteins. These motifs are normally short-sequence elements whose activity can be influenced by their secondary structure [7]. More recent data suggest that interactions can occur across classes [9] Upon binding to their cognate ephrin ligands, Eph receptors autophosphorylate and activate downstream signaling cascades (forward signaling). They do not possess catalytic activity themselves, both classes of ephrin ligands can activate signal transduction pathways after interaction with Eph receptors (reverse signaling) [10]

Methods

Results

Conclusion