The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development

Abstract MicroRNAs are important regulators of local protein synthesis during neuronal development. We investigated the dynamic regulation of microRNA production and found that the majority of the microRNA‐generating complex, consisting of Dicer, TRBP, and PACT, specifically associates with intracellular membranes in developing neurons. Stimulation with brain‐derived neurotrophic factor (BDNF), which promotes dendritogenesis, caused the redistribution of TRBP from the endoplasmic reticulum into the cytoplasm, and its dissociation from Dicer, in a Ca2+‐dependent manner. As a result, the processing of a subset of neuronal precursor microRNAs, among them the dendritically localized pre‐miR16, was impaired. Decreased production of miR‐16‐5p, which targeted the BDNF mRNA itself, was rescued by expression of a membrane‐targeted TRBP. Moreover, miR‐16‐5p or membrane‐targeted TRBP expression blocked BDNF‐induced dendritogenesis, demonstrating the importance of neuronal TRBP dynamics for activity‐dependent neuronal development. We propose that neurons employ specialized mechanisms to modulate local gene expression in dendrites, via the dynamic regulation of microRNA biogenesis factors at intracellular membranes of the endoplasmic reticulum, which in turn is crucial for neuronal dendrite complexity and therefore neuronal circuit formation and function.

. BDNF affects Dicer binding to TRBP and PACT and bi-directionally regulates the total levels of a few miRNAs.
A BDNF causes a decrease in the Dicer-TRBP interaction in co-IP experiments using a monoclonal mouse TRBP antibody or IgG control. Young cortical neurons were treated with BDNF or vehicle control for 20 min before lysis. The star depicts the right band for immunoprecipitated TRBP (43 kDa). B BDNF does not affect the interaction of cytoplasmic Dicer and TRBP. Cytoplasmic fractions of stimulated cortical neurons were isolated, and co-IP was performed using anti-TRBP antibody or control IgG. The star depicts the right band for immunoprecipitated TRBP (43 kDa). C BDNF does not affect global levels of microRNAs in small RNA sequencing. The average read counts (log 2 ) of miRNAs from three independent experiments were plotted on the x-axis for the control and y-axis for the BDNF-treated conditions. D BDNF stimulation bi-directionally regulates the levels of a few microRNAs in small RNA sequencing (P < 0.05, n = 3, t-test type 2, error bars are s.d.). E BDNF does not lead to changes in 5p versus 3p strand selection. The read count ratio (log 2 ) of 5p and 3p miRNAs, generated from the same precursor, was calculated from small RNA sequencing data. The averaged values for three independent experiments were plotted on the x-axis for the control and y-axis for the BDNF-treated conditions.
ª 2017 The Authors EMBO reports e44853 | 2017 Figure EV3. Short-term BDNF stimulation causes changes in the isomiR levels of many miRNAs.
Cortical neurons were treated with BDNF or vehicle control for 20 min, and isomiR levels were analyzed in small RNA sequencing. Shown are the miRNAs that have a significant change in the relative levels of at least one isomiR. The list is arranged in ascending order of the fold change of canonical isomiRs for each miRNA. Shown in red are members of the miR16 family, miR-16-5P, miR-15b-5p, and miR-15a-5p. Canonical isomiRs for each miRNA are depicted as "0". Numbers represent the trimming (negative) or addition (positive) of nucleotides to the 3 0 -or 5 0 -end of the canonical 5p or 3p isomiRs, respectively. Only templated nucleotide additions and trimmings, which might reflect Dicer-mediated cleavage, have been considered for analysis (P < 0.05, n = 3, t-test type 3).
EMBO reports e44853 | 2017 ª 2017 The Authors  Figure EV5. TRBP relocalization from the membrane to the cytoplasm is required for BDNF-induced dendritogenesis.
A Membrane-targeted GFP-TRBP (mGFP-TRBP) is present at the membrane fraction. HEK293T cells were transfected with 200 ng GFP, 400 ng GFP-TRBP, or 400 ng mGFP-TRBP, and sequential detergent extraction was performed 24 h later to isolate cytoplasmic and membrane fractions. B Distribution of cytoplasmic and membrane-associated GFP-TRBP in immunofluorescence of hippocampal neurons. Neurons were transfected at 4 DIV with dsRed and GFP-TRBP or mGFP-TRBP and fixed 3 days later. Note that GFP-TRBP is not exclusively cytoplasmic (scale bars are 10 lm). C Sholl analysis in BDNF-or control-treated hippocampal neurons expressing GFP, GFP-TRBP, or mGFP-TRBP. mGFP-TRBP blocks BDNF-induced dendritogenesis, whereas GFP-TRBP has a partial effect. The number of dendritic intersections is shown for each concentric circle, in increasing order of distance from the cell soma (n = 3, error bars; s.e.m.).