title: Assessing specific oligonucleotides and small molecule antibiotics for the ability to inhibit the CRD-BP-CD44 RNA interaction.
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.
authors.affiliation: Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
authors.forename: Dustin T
authors.initials: DT
authors.lastname: King
authors.affiliation: Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
authors.forename: Mark
authors.initials: M
authors.lastname: Barnes
authors.affiliation: Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
authors.forename: Dana
authors.initials: D
authors.lastname: Thomsen
authors.affiliation: Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
authors.forename: Chow H
authors.initials: CH
authors.lastname: Lee
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000617:Aminoglycosides / Q000737:chemistry / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D018960:Hyaluronan Receptors; C406144:IGF2BP1 protein, human; D016376:Oligonucleotides, Antisense; D010446:Peptide Fragments; D016601:RNA-Binding Proteins