ACCELERATED COMMUNICATION Control of Glutamate Receptor 2 (GluR2) Translational Initiation by Its Alternative 3 Untranslated Regions

Four major glutamate receptor 2 (GluR2) transcripts differing in size ( 4 and 6 kilobases) due to alternative 3 untranslated regions (UTRs), and also containing alternative 5 UTRs, exist in the brain. Both the long 5 UTR and long 3 UTR repress translation of GluR2 mRNA; repression by the 3 UTR is relieved after seizures. To understand the mechanism of translational repression, we used rabbit reticulocyte lysates as an in vitro translation system to examine the expression profiles of firefly reporter mRNAs bearing alternative combinations of GluR2 5 UTR and 3 UTR in the presence of inhibitors of either translational elongation or initiation. Translation of reporter mRNAs bearing the long GluR2 3 UTR was insensitive to low concentrations of the translation elongation inhibitors cycloheximide (0.7–70 nM) and anisomycin (7.5–750 nM), in contrast to a reporter bearing the short 3 UTR, which was inhibited. These data suggest that the rate-limiting step for translation of GluR2 mRNA bearing the long 3 UTR is not elongation. Regardless of the GluR2 UTR length, translation of all reporter mRNAs was equally sensitive to desmethyl-desamino-pateamine A (0.2–200 nM), an initiation inhibitor. Kasugamycin, which can facilitate recognition of certain mRNAs by ribosomes leading to alternative initiation, had no effect on translation of a capped reporter bearing both short 5 UTR and short 3 UTR, but increased the translation rate of reporters bearing either the long GluR2 5 UTR or long 3 UTR. Our findings suggest that both the long 5 UTR and long 3 UTR of GluR2 mRNA repress translation at the initiation step. AMPA receptors, a subfamily of ionotropic glutamate receptors mediating the majority of fast excitatory neurotransmission in the central nervous system (Dingledine et al., 1999), are ligand-gated ion channels formed by heteromeric or homomeric combinations of GluR1, GluR2, GluR3, and GluR4 subunits (Köhler et al., 1994; Rosenmund et al., 1998). Although most well known examples of gene regulation involve transcription, the efficiency of mRNA translation can be regulated in a transcript-specific manner by structural motifs residing in the 5 or 3 -UTR, alternate or additional 5 -UTR AUG codons (or their cognate short open reading frames), RNA binding proteins, or the nucleotide context of the initiator AUG (Gray and Wickens, 1998). Control of translation and trafficking of GluR2 subunits is involved in long-term synaptic potentiation (Isaac et al., 2007; Gainey et al., 2009). Multiple GluR2 transcripts exist in hippocampus that have alternative 5 and 3 -untranslated regions (UTRs) (Köhler et al., 1994; Myers et al., 1998; Irier et al., 2009). GluR2 mRNAs with at least two different 3 UTRs, long ( 2750 bases) and short ( 750 bases), exist in the brain (Köhler et al., 1994) but encode the same protein. GluR2 transcription is regulated by positive and negative regulatory elements in the promoter region(Myers et al., 1998; Huang et al., 1999). Translation is repressed by GU repeats located in the long 5 -UTR, and also by unknown elements in the long 3 UTR (Myers et al., 2004; Irier et al., 2009). Seizures reduce overall GluR2 mRNAs level but derepress the translation of GluR2 mRNAs bearing the long 3 UTRs in rat hippocampus (Irier et al., 2009). These findings suggest that GluR2 mRThis work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant R01-NS036604]. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.060343. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: AMPA, -amino-3-hydroxy-5-methylisoxazole-4-propionate; GluR, glutamate receptor; UTR, untranslated region; eIF, eukaryotic initiation factor; tRNA, transfer RNA; SS, flanked by short 5 UTR and short 3 UTR of GluR2; SL, flanked by short 5 UTR and long 3 UTR of GluR2; LS, flanked by long 5 UTR and short 3 UTR of GluR2; PCR, polymerase chain reaction; RLU, relative light units; DMDA-PatA, desmethyldesamino-pateamine A; IRES, internal ribosomal entry site. 0026-895X/09/7606-1145–1149$20.00 MOLECULAR PHARMACOLOGY Vol. 76, No. 6 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 60343/3540629 Mol Pharmacol 76:1145–1149, 2009 Printed in U.S.A. 1145 http://molpharm.aspetjournals.org/content/suppl/2009/09/30/mol.109.060343.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A ril 3, 2017 m oharm .aspeurnals.org D ow nladed from NAs with alternative combinations of GluR2 5 and 3 -UTRs are subject to transcript-specific regulation of translation, but the regulated step of translation is unknown. In general, eukaryotic mRNA translation occurs in four consecutive phases: initiation, elongation, termination, and ribosome recycling. In the initiation phase, a 43S preinitiation complex is formed by interaction of 40S ribosomal subunits with initiator methionyl transfer RNA (tRNA). This preinitiation ribosomal complex then binds to mRNA at the 5 cap structure (methylated GTP, m7GpppN) in a process facilitated by translation factor complex eIF4, and begins to scan through the 5 UTR, unwinding the secondary structure until it encounters the first eligible AUG codon, which is then oriented onto the P (peptidyl) site of the scanning ribosome. Once this start signal is recognized, the larger 60S subunit joins the 40S to form an 80S initiation complex that is ready to accept appropriate aminoacyl-tRNA into the A (aminoacyl) site on the ribosome, thus starting the elongation phase of translation (Kozak, 1989; Pestova et al., 2001; Sonenberg and Hinnebusch, 2009) . One approach to determining whether initiation or elongation is the rate-limiting step in protein expression is to treat cells with low concentrations of modulators of these processes (Lodish and Jacobson, 1972; Walden et al., 1981; Chen and Sarnow, 1995). If elongation is the rate-limiting step, then translation rate should be sensitive to a low concentration of elongation inhibitors. On the contrary, if translation of an mRNA is insensitive to low concentrations of translation elongation inhibitors or is sensitive to translation initiation modulators, then the rate-limiting step for such mRNAs is probably initiation. Taking this approach with rabbit reticulocyte lysates as an in vitro translation system, we report that translational repression caused by the long 5 UTR and, surprisingly, also the long 3 UTR is mediated at the initiation step. Materials and Methods GluR2 5 and 3 UTR Constructs and in Vitro Reporter mRNA Synthesis. The firefly luciferase reporter mRNAs bearing alternative GluR2 5 and 3 -UTRs were constructed using methods described previously (Irier et al., 2009). In brief, these constructs contain a firefly luciferase coding region flanked by alternative combinations of GluR2 5 and 3 -UTRs (see Fig. 1A; for Luciferase protein coding region, designated SS if flanked by short 5 UTR and short 3 UTR of GluR2, SL if flanked by short 5 UTR and long 3 UTR of GluR2, or LS if flanked by long 5 UTR and short 3 UTR of GluR2). These constructs were linearized at the 3 -end using the BstEII restriction site. In vitro synthesis of 5 -capped mRNAs from the linearized reporter constructs was performed using T3 RNA polymerase following the instructions provided in the T3 mMESSAGE mMACHINE (Ambion, Austin, TX). The resulting mRNAs were quantified and quality-checked in the RNA Nano Chip apparatus using a model 2100 Bioanalyzer (Agilent, Waldbronn, Germany). Reporter mRNAs were stored in RNase-free water as small aliquots of 100 M stocks at 80°C. As an additional control for obtaining luciferase reporter mRNAs with full-length GluR2 long 3 UTRs after the in vitro synthesis of reporter mRNAs, the reporter mRNAs bearing long GluR2 3 UTRs (SL) were reverse-transcribed to cDNAs using Thermoscript reverse transcriptase (Invitrogen, Carlsbad, CA) and amplified using primer pairs specific to short (3022 forward and 3119 reverse), long proximal (3891 forward and 4015 reverse), and long distal (4403 forward and 4597 reverse) regions of GluR2 3 UTR. The number of PCR cycles required to reach threshold were compared for the three primer pairs. In Vitro Translation of Reporters in Rabbit Reticulocyte Lysates. Commercially available rabbit reticulocyte lysates were used according to the instructions provided with Retic Lysate IVT (Applied Biosystems, Foster City, CA). In brief, a 50l final translation assay mix was assembled in a 0.5l thin-walled PCR reaction tube on ice, and the components were added in the following order: drug or (vehicle), 34 l of rabbit reticulocyte lysate, 20 Translation Mix(-met), 1 mM methionine, 0.8 U of RNase inhibitor, reporter mRNA, and RNase-free water. The translation mix was incubated at 30°C for up to 40 min using a digitally monitored heat block. A 5l sample from the final translation mix was removed at 10-min intervals and flash frozen in a 96-well plate (Microlite white flat-bottomed plates; Thomas Scientific, Swedesboro, NJ) that was placed on dry ice. The luciferase activity, defined as recorded luminescence units (RLU) measured at 570 nm, was measured from the individual wells using the luciferase assay substrate (Dual-Luciferase Reporter Assay System; Promega, Madison, WI). The RLU from the 5l reticulocyte samples without reporter mRNAs (0.05–0.5 RLU) constituted background, which was consistent for each 96-well plate used. The lowest experimental sample RLUs at 20 min were at least 100-fold Fig. 1. In vitro transcription of luciferase reporters bearing alternative combinations of GluR2 UTRs. A, schematic illustration of firefly luciferase reporter mRNAs bearing alternative combinations of GluR2, 5 and 3 UTRs. The luciferase coding region is common to all reporters. The dark gray box indicates the position of GU repeats on the long 5 UTR (Myers et al., 2004). B, the quality and amount of each in vivo-transcribed firefly reporter were evaluated with an Agilent 2100 Bioanalyzer, which produces a gel-like image. The expected sizes of the transcripts is SS, 2760; SL, 4710; and LS, 3140 base pairs. C, the activity of luciferase expressed from the firefly reporter mRNAs bearing the short 5 and long 3 -UTRs of GluR2 (SL) was proportional to both mRNA amount and incubation time in rabbit reticulocyte lysate translation mix. 1146 Irier et al. at A PE T Jornals on A ril 3, 2017 m oharm .aspeurnals.org D ow nladed from higher than background. To determine reporter mRNA stability, each firefly reporter mRNA was extracted from the rabbit reticulocyte lysates at time 0 and 40 min using a standard phenol-chlorophorm method. The extracted mRNA was dissolved in a 20l in vitro reverse transcription reaction that contained Thermoscript reverse transcriptase (Invitrogen), and the reaction was run for 50 min at 50°C. The resulting cDNAs were quantified by quantitative real-time PCR analysis (SyberGreen Supermix; Applied Biosystems) using primer sets that amplified UTR-specific regions along with standard dilutions of the luciferase reporter cDNA. Drugs Used. DMDA-PatA was kindly made available to us by Dr. Daniel Romo (Department of Chemistry, Texas A&M University, College Station, TX). Cycloheximide and anisomycin were purchased from Sigma-Aldrich (St. Louis, MO). Kasugamycin was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). DMDA-PatA and anisomycin were dissolved in dimethyl sulfoxide. Statistics. For the statistical analyses of the data sets for each drug treatment shown in Fig. 2, the one-way analysis of variance was used with the post test Bonferroni multiple comparison test.