Post-transcriptional regulation of mu-opioid receptor: role of the RNA-binding proteins heterogeneous nuclear ribonucleoprotein H1 and F.

Classical opioids have been historically used for the treatment of pain and are among the most widely used drugs for both acute severe pain and long-term pain. Morphine and endogenous mu-opioid peptides exert their pharmacological actions mainly through the mu-opioid receptor (MOR). However, the expression of opioid receptor (OR) proteins is controlled by extensive transcriptional and post-transcriptional processing. Previously, the 5'-untranslated region (UTR) of the mouse MOR was found to be important for post-transcriptional regulation of the MOR gene in neuronal cells. To identify proteins binding to the 5'-UTR as potential regulators of the mouse MOR gene, affinity column chromatography using 5'-UTR-specific RNA oligonucleotides was performed using neuroblastoma NS20Y cells. Chromatography was followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified two heterogeneous ribonucleoproteins (hnRNPs) that bound to RNA sequences of interest: hnRNP H1 and hnRNP F. Binding of these proteins to the RNA region was M4-region sequence-specific as confirmed by Western-blot analysis and RNA supershift assay. Furthermore, a cotransfection study showed that the presence of hnRNP H1 and F resulted in repressed expression of the mouse MOR. Our data suggest that hnRNP H1 and F can function as repressors of MOR translation dependent on the M4 (-75 to -71 bp upstream of ATG) sequences. We demonstrate for the first time a role of hnRNPs as post-transcriptional repressors in MOR gene regulation.