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miR-501-3p mediates the activity-dependent regulation of the expression of AMPA receptor subunit GluA1.

Hu Z, Zhao J, Hu T, Luo Y, Zhu J, Li Z - J. Cell Biol. (2015)

Bottom Line: We used the 3' untranslated region of Gria1, which encodes the AMPA receptor subunit GluA1, to pull down miRNAs binding to it and analyzed these miRNAs using next-generation deep sequencing.Among the identified miRNAs, miR-501-3p is also a computationally predicted Gria1-targeting miRNA.These findings elucidate a miRNA-mediated mechanism for activity-dependent, local regulation of AMPAR expression in dendrites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unit on Synapse Development and Plasticity, National Institute of Mental Health, and Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.

ABSTRACT
The number of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in synapses determines synaptic strength. AMPAR expression can be regulated locally in dendrites by synaptic activity. The mechanisms of activity-dependent local regulation of AMPAR expression, however, remain unclear. Here, we tested whether microRNAs (miRNAs) are involved in N-methyl-D-aspartate (NMDA) receptor (NMDAR)-dependent AMPAR expression. We used the 3' untranslated region of Gria1, which encodes the AMPA receptor subunit GluA1, to pull down miRNAs binding to it and analyzed these miRNAs using next-generation deep sequencing. Among the identified miRNAs, miR-501-3p is also a computationally predicted Gria1-targeting miRNA. We confirmed that miR-501-3p targets Gria1 and regulates its expression under physiological conditions. The expression of miR-501-3p and GluA1, moreover, is inversely correlated during postnatal brain development. miR-501-3p expression is up-regulated locally in dendrites through the NMDAR subunit GluN2A, and this regulation is required for NMDA-induced suppression of GluA1 expression and long-lasting remodeling of dendritic spines. These findings elucidate a miRNA-mediated mechanism for activity-dependent, local regulation of AMPAR expression in dendrites.

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Identification of Gria1 targeting miRNAs. Small RNAs isolated from the hippocampus of mice (17 d old) were incubated with Gria1 3′ UTR-bound beads for pull-down of Gria1 binding miRNAs. (A) Schematic illustration of the pull-down assay. (B) Overlap of miRNAs identified by pull-down and those predicted by TargetScan to bind to Gria1 3′ UTR at conserved binding sites.
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fig1: Identification of Gria1 targeting miRNAs. Small RNAs isolated from the hippocampus of mice (17 d old) were incubated with Gria1 3′ UTR-bound beads for pull-down of Gria1 binding miRNAs. (A) Schematic illustration of the pull-down assay. (B) Overlap of miRNAs identified by pull-down and those predicted by TargetScan to bind to Gria1 3′ UTR at conserved binding sites.

Mentions: The level of GluA1 protein is reduced upon NMDAR activation (Grooms et al., 2006). To determine whether miRNAs are involved in this process, we first screened for miRNAs that target the GluA1 encoding gene Gria1. The mouse Gria1 mRNA can be targeted by ∼200 miRNAs through both conserved and nonconserved binding sites as predicted by miRNA target prediction tools (such as TargetScan). Computational prediction of miRNA targets, however, has a high false positive error rate (Liu et al., 2014). To avert this problem, we experimentally identified miRNAs targeting Gria1 by using the 3′ UTR of Gria1 mRNA to pull down miRNAs that bind to it (Fig. 1 A). Mouse Gria1 3′ UTR were transcribed in vitro, and the resulting mRNAs were biotinylated at their 3′ ends and immobilized to avidin-agarose beads. The RNA-avidin beads were used to pull down miRNAs isolated from mouse brains. Both eluted and input RNAs from the pull-down assay were analyzed by next-generation deep sequencing. 43 miRNAs that were enriched >10-fold by pull-down were considered candidate Gria1-targeting miRNAs (Fig. 1 B). We then searched the mouse Gria1’s 3′ UTR for miRNA binding sites using TargetScan 6.2. To reduce the false discovery rate, only conserved binding sites were selected. Among the 43 miRNAs identified by the pull-down assay, only miR-501-3p is predicted by TargetScan to bind to Gria1 3′ UTR at a conserved binding site (Fig. 1 B). miR-501-3p, therefore, is our experimentally and computationally identified miRNA-targeting Gria1.


miR-501-3p mediates the activity-dependent regulation of the expression of AMPA receptor subunit GluA1.

Hu Z, Zhao J, Hu T, Luo Y, Zhu J, Li Z - J. Cell Biol. (2015)

Identification of Gria1 targeting miRNAs. Small RNAs isolated from the hippocampus of mice (17 d old) were incubated with Gria1 3′ UTR-bound beads for pull-down of Gria1 binding miRNAs. (A) Schematic illustration of the pull-down assay. (B) Overlap of miRNAs identified by pull-down and those predicted by TargetScan to bind to Gria1 3′ UTR at conserved binding sites.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384731&req=5

fig1: Identification of Gria1 targeting miRNAs. Small RNAs isolated from the hippocampus of mice (17 d old) were incubated with Gria1 3′ UTR-bound beads for pull-down of Gria1 binding miRNAs. (A) Schematic illustration of the pull-down assay. (B) Overlap of miRNAs identified by pull-down and those predicted by TargetScan to bind to Gria1 3′ UTR at conserved binding sites.
Mentions: The level of GluA1 protein is reduced upon NMDAR activation (Grooms et al., 2006). To determine whether miRNAs are involved in this process, we first screened for miRNAs that target the GluA1 encoding gene Gria1. The mouse Gria1 mRNA can be targeted by ∼200 miRNAs through both conserved and nonconserved binding sites as predicted by miRNA target prediction tools (such as TargetScan). Computational prediction of miRNA targets, however, has a high false positive error rate (Liu et al., 2014). To avert this problem, we experimentally identified miRNAs targeting Gria1 by using the 3′ UTR of Gria1 mRNA to pull down miRNAs that bind to it (Fig. 1 A). Mouse Gria1 3′ UTR were transcribed in vitro, and the resulting mRNAs were biotinylated at their 3′ ends and immobilized to avidin-agarose beads. The RNA-avidin beads were used to pull down miRNAs isolated from mouse brains. Both eluted and input RNAs from the pull-down assay were analyzed by next-generation deep sequencing. 43 miRNAs that were enriched >10-fold by pull-down were considered candidate Gria1-targeting miRNAs (Fig. 1 B). We then searched the mouse Gria1’s 3′ UTR for miRNA binding sites using TargetScan 6.2. To reduce the false discovery rate, only conserved binding sites were selected. Among the 43 miRNAs identified by the pull-down assay, only miR-501-3p is predicted by TargetScan to bind to Gria1 3′ UTR at a conserved binding site (Fig. 1 B). miR-501-3p, therefore, is our experimentally and computationally identified miRNA-targeting Gria1.

Bottom Line: We used the 3' untranslated region of Gria1, which encodes the AMPA receptor subunit GluA1, to pull down miRNAs binding to it and analyzed these miRNAs using next-generation deep sequencing.Among the identified miRNAs, miR-501-3p is also a computationally predicted Gria1-targeting miRNA.These findings elucidate a miRNA-mediated mechanism for activity-dependent, local regulation of AMPAR expression in dendrites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unit on Synapse Development and Plasticity, National Institute of Mental Health, and Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.

ABSTRACT
The number of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in synapses determines synaptic strength. AMPAR expression can be regulated locally in dendrites by synaptic activity. The mechanisms of activity-dependent local regulation of AMPAR expression, however, remain unclear. Here, we tested whether microRNAs (miRNAs) are involved in N-methyl-D-aspartate (NMDA) receptor (NMDAR)-dependent AMPAR expression. We used the 3' untranslated region of Gria1, which encodes the AMPA receptor subunit GluA1, to pull down miRNAs binding to it and analyzed these miRNAs using next-generation deep sequencing. Among the identified miRNAs, miR-501-3p is also a computationally predicted Gria1-targeting miRNA. We confirmed that miR-501-3p targets Gria1 and regulates its expression under physiological conditions. The expression of miR-501-3p and GluA1, moreover, is inversely correlated during postnatal brain development. miR-501-3p expression is up-regulated locally in dendrites through the NMDAR subunit GluN2A, and this regulation is required for NMDA-induced suppression of GluA1 expression and long-lasting remodeling of dendritic spines. These findings elucidate a miRNA-mediated mechanism for activity-dependent, local regulation of AMPAR expression in dendrites.

Show MeSH
Related in: MedlinePlus