Abstract
Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3′UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862–3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862–3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions.
Highlights
The coding region determinant-binding protein (CRD-BP), commonly known as insulin-like growth factor II mRNA-binding protein 1 (IMP1), is a member of the conserved VICKZ family of RNA-binding proteins that are characterized by the presence of two N-terminal RNA-recognition motifs (RRMs) followed by four C-terminal KH [hnRNP K-homology] domains [1,2,3,4]
Further efforts toward finding inhibitory molecules are required because such compounds would have great therapeutic potential and can assist in elucidating the mechanism whereby CRD-BP binds to its wide selection of RNA targets
We describe the characterization of the CRD-BP-CD44 RNA interaction and evaluate a panel of antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction
Summary
The coding region determinant-binding protein (CRD-BP), commonly known as insulin-like growth factor II mRNA-binding protein 1 (IMP1), is a member of the conserved VICKZ family of RNA-binding proteins that are characterized by the presence of two N-terminal RNA-recognition motifs (RRMs) followed by four C-terminal KH [hnRNP (heterogenous nuclear ribonucleoprotein) K-homology] domains [1,2,3,4]. CRD-BP has about 95% protein sequence identity to the chicken zipcode-binding protein 1 (ZBP1) [2] that associates with b–actin mRNA [5,6]. Mice deficient in IMP1 developed dwarfism and exhibited severe impairment in intestinal phenotype at early birth [7]. This is consistent with the observation that CRD-BP is abundantly expressed in fetal tissues but not in normal adult tissues [8]. The study suggests that this RNA-binding protein plays a significant role in intestinal development of the fetus. Studies in Drosophila melanogaster, Xenopus laevis and mammalian neuronal cells have provided evidence for the role of IMP1 in nervous system development [1]. The ability of IMP1 to promote neurite outgrowth and branching is predominantly due to its role in controlling specific mRNA localization and translation, in particular the b–actin mRNA [1]
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