Research ArticleNEUROSCIENCE

BDNF-induced local translation of GluA1 is regulated by HNRNP A2/B1

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Science Advances  20 Nov 2020:
Vol. 6, no. 47, eabd2163
DOI: 10.1126/sciadv.abd2163
  • Fig. 1 BDNF-induced local translation of GluA1.

    (A) Representative hippocampal primary neuron images and close-ups of Puro-PLA signal for newly synthesized GluA1 without puromycin labeling (Control), with 15-min labeling of Puro alone (Puro), or with the protein synthesis inhibitor anisomycin (Puro+Aniso) or BDNF (Puro+BDNF). Scale bars, 25 μm (overview) and 15 μm (close-ups). (B) Summary graph of normalized PLA intensities of hippocampal primary neurons. The PLA intensity of control was set to 1. Data represent means ± SEM (n = 8, *P < 0.05, **P < 0.01). (C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis for GluA1 mRNA levels of hippocampal neurons after BDNF treatment. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA level was used for normalization (n = 5). n.s., not significant.

  • Fig. 2 Cap-independent translation of GluA1 mRNA.

    (A) Schematic diagram of the bicistronic reporter plasmid containing the GluA1 5′UTR. The full length of GluA1 5′UTR or the reverse sequence of the GluA1 5′UTR was inserted between the Rluc and Fluc sequences of the pRF plasmid. (B) Neuro2a cells were transfected with bicistronic reporters, and the efficiency of cap-independent translation of each plasmid is shown. The ratio of the control pRF vector was set to 1. Data represent means ± SEM (n = 5, ****P < 0.0001). (C and D) Representative images of hippocampal neurons transfected with the control fluorescent bicistronic reporter (C) and reporter harboring the GluA1 5′UTR and 3′UTR (D). Scale bar, 20 μm.

  • Fig. 3 BDNF activates IRES-mediated translation of GluA1 mRNA.

    (A) Representative images of hippocampal neurons transfected with fluorescent bicistronic reporters after treatment with rapamycin, BDNF, or both. Scale bar, 50 μm. (B) Summary graph illustrating the fluorescence intensity of DsRed and GFP shown in (A). Intensities of samples were normalized with control samples. Data represent means ± SEM (n = 7 to 10 neurons per condition from three independent experiments, *P < 0.05). n.s., not significant.

  • Fig. 4 Determination of the cis-acting element in IRES-mediated translation of GluA1.

    (A) Schematic diagram of the bicistronic reporter containing the serially deleted GluA1 5′UTR. (B) Neuro2a cells were transfected with serial deletion plasmids, and a luciferase assay was performed. The efficiency of cap-independent translation of each plasmid is shown. The ratio of the control pRF vector was set to 1. Data represent means ± SEM (n = 5, **P < 0.01, ****P < 0.0001). (C to E) Representative images of hippocampal neurons transfected with control fluorescent bicistronic reporter (C), reporter containing the full-length GluA1 5′UTR (D), or reporter harboring a serially deleted GluA1 5′UTR (E). Images were visualized by super-resolution structured illumination microscopy. Dendrites and dendritic spines were outlined by white line. Scale bar, 1 μm.

  • Fig. 5 HNRNP A2/B1 interacts with the GluA1 5′UTR.

    (A) Various RNA binding proteins that bound to the GluA1 5′UTR were determined using an in vitro binding assay. The biotin-conjugated GluA1 5′UTR was incubated with Neuro2a cytoplasmic cell extracts and subjected to immunoblotting using specific antibodies. A biotin-unconjugated GluA1 5′UTR was used as competitor. (B) Confirmation of the interaction between HNRNP A2/B1 and the GluA1 5′UTR. Biotin-conjugated full-length and serially deleted forms of the GluA1 5′UTR were incubated with the crude synaptosomal fraction from the mouse hippocampus and subjected to immunoblotting using hnRNP A2/B1 antibody. (C) Cis-acting region of GluA1 5′UTR preferentially interacts with eukaryotic initiation factor 3B, HNRNP A2/B1, and RPS25. Biotin-conjugated cis-acting region and truncated forms of GluA1 5′UTR were mixed with Neuro2a cytoplasmic cell lysates and followed by immunoblotting using specific antibodies.

  • Fig. 6 HNRNP A2/B1 regulates BDNF-induced GluA1 local translation.

    (A to D) Representative images of newly translated GluA1 after puromycin labeling in hippocampal neurons transfected with a control shRNA (A and C) or shRNA targeting Hnrnpa2b1 (B and D). Neurons stimulated with BDNF are shown in (C) and (D). Insets of GluA1–Puro-PLA images are enlarged on the right. Scale bar, 20 μm. (E and F) Summary graphs showing PLA signal intensity in somata and dendrites of untransfected neurons or those expressing the control shRNA (E) or Hnrnpa2b1 targeting shRNA (F). The fluorescence intensity of untransfected neurons without BDNF stimulation was set to 1. Data represent means ± SEM (n = 11 to 15 neurons per condition from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001), n.s., not significant. (G) Representative images showing dendrites of control or HNRNP A2/B1–depleted neurons. Scale bar, 20 μm. (H) Summary graph of dendritic spine density obtained in neurons transfected with a control shRNA or Hnrnpa2b1 targeting shRNA. Data represent means ± SEM (n = 9 to 15 neurons per condition from three independent experiments, *P < 0.05).

Supplementary Materials

  • Supplementary Materials

    BDNF-induced local translation of GluA1 is regulated by HNRNP A2/B1

    Youngseob Jung, Ji-Young Seo, Hye Guk Ryu, Do-Yeon Kim, Kyung-Ha Lee, Kyong-Tai Kim

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