Translational regulation of GluR2 mRNAs in rat hippocampus by alternative 3′ untranslated regions

Abstract

The glutamate receptor 2 (GluR2) subunit determines many of the functional properties of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate subtype of glutamate receptor. The roles of untranslated regions (UTRs) in mRNA stability, transport, or translation are increasingly recognized. The 3′ end of the GluR2 transcripts are alternatively processed to form a short and long 3′UTR, giving rise to two pools of GluR2 mRNA of 4 and 6 kb in length, respectively, in the mammalian brain. However, the role of these alternative 3′UTRs in GluR2 expression has not been reported. We demonstrate that in the cytoplasm of rat hippocampus, native GluR2 mRNAs bearing the long 3′UTR are mostly retained in translationally dormant complexes of ribosome‐free messenger ribonucleoprotein (mRNP), whereas GluR2 transcripts bearing the short 3′UTR are predominantly associated with actively translating ribosomes. One day after pilocarpine‐induced status epilepticus (SE), the levels of both long and short GluR2 transcripts were markedly decreased in rat hippocampus. However, GluR2 mRNAs bearing the long 3′‐UTRs were shifted from untranslating mRNP complexes to ribosome‐containing complexes after SE, pointing to a selective translational derepression of GluR2 mRNA mediated by the long 3′UTR. In Xenopus oocytes, expression of firefly luciferase reporters bearing alternative GluR2 3′UTRs confirmed that the long 3′UTR is sufficient to suppress translation. The stability of reporter mRNAs in oocytes was not significantly influenced by alternative 5′ or 3′UTRs of GluR2 over the time period examined. Overall, our findings that the long 3′UTR of GluR2 mRNA alone is sufficient to suppress translation, and the evidence for seizure‐induced derepression of translation of GluR2 via the long 3′UTR strongly suggests that a regulatory signaling mechanism exists that differentially targets GluR2 transcripts with alternative 3′UTRs.

Distinct 3′UTRs differentially regulate activity-dependent translation of brain-derived neurotrophic factor (BDNF)

“…Animals were treated in accordance with National Institutes of Health regulations under the approval of the Emory University Institutional Animal Care and Use Committee. Pilocarpine reliably induced stage V seizures in adult male Sprague-Dawley rats and C57 Bl/6 mice (Charles River Labs), characterized by distinct motor behaviors including forelimb clonus, loss of postural control, rearing, and falling (35). Animals presenting these behaviors with increased seizure intensity, duration and frequency 20 to 40 min after the injection of pilocarpine were declared to be in SE, and the initial onset of stage V seizure was used to mark the start point of SE.…”

“…Hippocampi were homogenized in a gradient buffer (20 mM Tris, pH 7.5, 100 mM KCl, 5 mM MgCl 2 ) containing 100 μg/mL cycloheximide to arrest polyribosome migration and then lysed with 1% Triton X-100. Cytoplasmic extracts were fractionated on 15% to 45% (wt/vol) sucrose gradient and total RNA was extracted as previously described (12,35). To dissociate polyribosomes, EDTA-treated lysate was centrifuged on a parallel gradient containing 1 mM EDTA.…”

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

“…Translation is repressed by GU repeats located in the long 5Ј-UTR, and also by unknown elements in the long 3ЈUTR (Myers et al, 2004;Irier et al, 2009). Seizures reduce overall GluR2 mRNAs level but derepress the translation of GluR2 mRNAs bearing the long 3ЈUTRs in rat hippocampus (Irier et al, 2009). These findings suggest that GluR2 mR-NAs with alternative combinations of GluR2 5Ј-and 3Ј-UTRs are subject to transcript-specific regulation of translation, but the regulated step of translation is unknown.…”

“…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 5Ј-UTR, and also by unknown elements in the long 3ЈUTR (Myers et al, 2004;Irier et al, 2009). Seizures reduce overall GluR2 mRNAs level but derepress the translation of GluR2 mRNAs bearing the long 3ЈUTRs in rat hippocampus (Irier et al, 2009).…”

“…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 5Ј-and 3Ј-untranslated regions (UTRs) (Köhler et al, 1994;Myers et al, 1998;Irier et al, 2009). GluR2 mRNAs with at least two different 3ЈUTRs, long (ϳ2750 bases) and short (ϳ750 bases), exist in the brain (Köhler et al, 1994) but encode the same protein.…”