The Nuclear Transcription Factor RARα Associates with Neuronal RNA Granules and Suppresses Translation

Abstract

All-trans-retinoic acid stimulates dendritic growth in hippocampal neurons within minutes by activating mitogen-activated protein kinase and mTOR and increasing dendritic translation of calcium calmodulin-dependent protein kinase II ␣ and the ␣-amino-3-hydroxyl-5-methyl-4-isoxazole propionate receptor subunit GluR1. Hippocampal neurons express RAR␣ in dendrites, and knocking down RAR␣ prevents all-trans-retinoic acid effects on dendritic growth. Here we show, by liquid chromatography/mass spectrometry analysis of immunoaffinity isolates of hippocampal neurons, that RAR␣ partners with many RNA-binding proteins and translation factors conveyed in dendritic RNA transport granules, including the purine-rich element-binding protein, Pur ␣. The interaction of RAR␣ with Pur ␣, an RNA-binding protein required for dendritic RNA transport, and other RNA-binding proteins was confirmed by tandem affinity purification. Confocal microscopy confirmed localization of neuronal RAR␣ in dendritic RNA granules with Pur ␣ and FMRP (the fragile ؋ mental retardation protein). Hippocampal RAR␣ also associates with mRNA, e.g. encoding GluR1 and calcium calmodulin-dependent protein kinase II ␣. Consistent with a granule function of conveying translationally silenced mRNA, RAR␣ inhibits translation initiation, independent of 7-methylguanylate cap or poly(A) tail, and prompts mRNA redistribution to silencing ribonucleoprotein particles. These data afford a mechanism for rapid stimulation of dendritic growth by all-trans-retinoic acid and reveal that the liganddependent transcription factor RAR␣ also regulates translation.The developed nervous system relies on atRA 2 for synaptic plasticity that underlies hippocampus-dependent spatial learning (1, 2). Restricting the dietary atRA precursor retinol (vitamin A) or genetically impairing atRA signaling impairs hippocampus-dependent learning (3-6). Retinoid-compromised rodents err markedly more frequently in tests related to hippocampal function, such as the radial arm and Morris water mazes, and have diminished long term potentiation and abolished long term depression. Conversely, atRA dosing reduces frequency of relational memory errors in older mice relative to young mice (7).Synaptic plasticity relies on protein synthesis in dendrites (8, 9). Large ribonucleoparticles or RNA granules convey translationally silenced mRNA from neuronal cell bodies to dendrites. These RNA encode diverse classes of postsynaptic proteins, including ionotropic glutamate receptors and kinases such as CaMKII ␣ (10). Synaptic activity triggers translation of dendritic mRNA to support rapid synaptic structure and efficacy modifications (10, 11). For example, the ␣-amino-3-hydroxyl-5-methyl-4-isoxazole propionate receptor subunits GluR1 and GluR2 insert into synaptic membranes after local translation to enhance the number, strength, and stability of synapses (12).atRA stimulates dendritic growth in primary mouse hippocampal neurons; siRNA knockdown and small hairpin RNA knockdown indicate a central function for RAR␣ in...

Retinoic acid-gated sequence-specific translational control by RARα

“…Our results are partly consistent with another study suggesting that RAR␣ is an RNA-binding protein (31). This study, however, postulates that RAR␣ binds mRNAs in a sequence-independent manner via its DNA-binding domain.…”

Drosophila Pur-α binds to trinucleotide-repeat containing cellular RNAs and translocates to the early oocyte

“…Indeed, inhibition of translation by Pur-α has been observed in vitro 34 and Pur-α was found associated with translational repressors in cells. 35 The absence of obvious dominant-negative effects for our GFP-Pur-α mutants with reduced RNA binding suggests that no essential factors are titrated away from the transport complexes, consistent with the notion that the interactions with Pur-α are mediated by RNA.…”

Multiple Retinol and Retinal Dehydrogenases Catalyze All-trans-retinoic Acid Biosynthesis in Astrocytes

“…Hippocampus neurons respond to atRA via retinoic acid receptor ␣ by quickly increasing dendrite outgrowth through a novel mechanism of regulating translation to increase CamKII kinase and GluR1 (12,17). The source of atRA in the hippocampus had not been established, whether from neurons, astrocytes, or more distance sources (e.g.…”

“…RNA concentration was determined by absorption at 260 nm; the 260/280 nm ratio was Ͼ1.9. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was done using oligo(dT) [12][13][14][15][16][17][18] as primer and Superscript II reverse transcriptase (Invitrogen) in a 20-l reaction mixture. The cDNA was diluted and amplified with Taq polymerase at the following conditions: initial denaturing step at 93°C for 3 min followed by 30 -35 cycles of denaturation (93°C, 30 s), annealing (60°C, 30 s), and extension (72°C, 30 s), then further extension at 72°C for 10 min.…”

“…Molecular, cellular, and behavioral studies confirm that central nervous system development and function rely on atRA (9 -11). atRA functions in the nervous system via the nuclear RA hormone receptors, to regulate both transcription and translation (12)(13)(14). For example, disrupting atRA signaling by knocking out retinoic acid receptor ␤ severely compromises performance in the Morris water maze test, commonly used to evaluate hippocampus-dependent spatial learning in rodents (15).…”