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

Abstract

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.

Fig. 1

Schematic representation of the mouse MOR 5′-UTR and toeprinting analysis. a The mouse MOR 5′-UTR contains the proximal promoter and three uAUGs. The mouse MOR translation initiation site is indicated by +1. Four transcription initiation sites (−291, −284, −276, and −268 bp from translational initiation site) are indicated by the arrow. b Toeprint analyses of initiation of uAUGs and unidentified bands. Synthetic RNA transcripts (100 ng) were used to program translation mixtures derived from RRL (Promega). Mouse MOR-LUC transcripts containing all three uORFs (i.e., wild-type; uAUG (+); or mutated at all three uAUGs [uAUG (−)]) were incubated at 30°C for 15 min in micrococcal nuclease-treated RRL. Radiolabeled mToe primer was used for primer extension analyses and for sequencing of the uAUG (+) templates. The sequence of the template can be directly deduced by 5′–3′ sequence reads from top to bottom. M Dephosphorylated ΦX174 HinfI Markers (Promega); arrow position of unidentified bands; open arrowhead toeprint of each uAUG and main AUG. c Unidentified bands were calculated by molecular weight marker and sequencing ladder. The unknown bands consisted of several G sequences (M1–M4)

Fig. 2

Translation of the mouse MOR gene is controlled by M4 mutation. a Schematic representation of reporter constructs with wild-type and mutated mouse MOR 5′-UTRs with M mutations (M1–M4). Vertical dotted lines represent ATGs converted to ACGs by point mutations; circled X’s indicate each M sequence mutation as described in Materials and methods. b Transient transfection of each mutant construct in NS20Y cells. After transfection, cells were trypsinized and half were used for luciferase and β-galactosidase activity assays, while half were used for RNA extraction and transcript quantification. Relative LUC activity and mRNA levels were determined as the ratio of LUC/β-gal and LUC/LacZ as described in Materials and methods. The error bars indicate the standard errors of triplicate LUC assays

Fig. 3

Schematic representation of the procedure for one-step purification of RNA binding proteins. a RNA oligonucleotides containing wild M4 and mutated M4 (mM4) nucleotide sequences with or without biotin label. b Outline of the modified one-step purification of RBPs using an affinity column. RNA oligonucleotides biotinylated on the 5′- or 3′-terminus were used as affinity particles. Cytosolic proteins were added to the affinity particles, incubated, and washed. Proteins bound to the particles were released by heating in SDS sample buffer. Competitor experiments to eliminate nonspecific binding (empty stars) and to identify specific binding (filled star) were performed by preincubating the cytosolic proteins with a two-fold excess of nonbiotinylated RNAs (M4-30 or mM4-30) as competitors prior to affinity binding. c Coomassie-stained gel of RNA binding proteins purified from NS20Y cytosolic extracts with competitor. The competitor RNAs M4-30 and mM4-30 are shown in panel a

Fig. 4

Simply Blue safe-stained 2-DE images of RBPs purified using an affinity column. Purified samples were separated on pH 3–10 IPG strips followed by separation by 12% SDS-PAGE. Competitor assay is shown in a and c; sample assay shown in b. Molecular weight markers are indicated on the left, and PI values are indicated the bottom. Indicated arrow spots were subjected to analysis by MALDI-TOF mass spectrometry and bioinformatics. Detailed information on each spot is listed in Table 1

Fig. 5

Identification of hnRNP H1 and F proteins as M4 binding proteins using mM4-30 competitor. Western-blot analysis of purified RBPs performed with anti-hnRNP H1 (a) and anti-hnRNP F (b) antibodies. HnRNP H1 (a) and hnRNP F (b) proteins level were measured in NS20Y cells by Western blotting after purification. Intensities of each signal were analyzed by ImageQuant 5.2 software

Fig. 6

Interaction of hnRNP H1 and F with M4 RNA derived from the mouse MOR 5′-UTR. a The mouse MOR M4-30 and mutated M4-30 RNA sequences. b HnRNP H1 and F proteins radiolabeled in vitro with [S³⁵]-methionine (lanes 1 and 2, respectively). c REMSAs performed with both RNA sequences and in vitro-labeled proteins. Lanes 1 and 5 M4-30 RNA nucleotide without antibody; lanes 2 and 6 mM4-30 RNA nucleotide without antibody; lanes 3 and 4 anti-hnRNP H1; lanes 7 and 8 anti-hnRNP F. The protein–RNA complexes are indicated by the arrow. d REMSAs were performed using ³²P-labeled M4-30 as a probe with non-labeled in vitro translated proteins. Lane 1 probe alone, lane 2 reticulocyte (RBC) without antibody, lane 3 no added antibody, lane 4 self-competitor without antibody, lane 5 anti-hnRNP H1 or F, lane 6 preimmune serum (PI). The protein–RNA complexes are indicated by the arrow; the empty arrow head indicates non-specific binding between RRL and RNA

Fig. 7

Mouse MOR expression levels in hnRNP H1- and F-transfected NS20Y cells. a Schematic representation of the mouse MOR 5′-UTR containing the reporter construct shown previously [15]. b Graphic representation of relative luciferase activity determined by luciferase assay (Promega) of the constructs shown in a with 2–4 μg of RBP expression constructs. Relative LUC activity and mRNA levels were determined as the ratio of LUC/β-gal and LUC/LacZ as described in the Materials and methods section. The error bars indicate the standard errors of triplicate LUC assays