The conserved microRNA miR-34 regulates synaptogenesis via coordination of distinct mechanisms in presynaptic and postsynaptic cells

Elizabeth M Mcneill,Tudor A Fulga,Todd F Deluca,Alicia Taylor,Leslie C Griffith,Chloe Warinner,Stephen Alkins,Hansine Heggeness,Dennis P Wall,David Van Vactor

doi:10.1038/s41467-020-14761-8

Abstract

Micro(mi)RNA-based post-transcriptional regulatory mechanisms have been broadly implicated in the assembly and modulation of synaptic connections required to shape neural circuits, however, relatively few specific miRNAs have been identified that control synapse formation. Using a conditional transgenic toolkit for competitive inhibition of miRNA function in Drosophila, we performed an unbiased screen for novel regulators of synapse morphogenesis at the larval neuromuscular junction (NMJ). From a set of ten new validated regulators of NMJ growth, we discovered that miR-34 mutants display synaptic phenotypes and cell type-specific functions suggesting distinct downstream mechanisms in the presynaptic and postsynaptic compartments. A search for conserved downstream targets for miR-34 identified the junctional receptor CNTNAP4/Neurexin-IV (Nrx-IV) and the membrane cytoskeletal effector Adducin/Hu-li tai shao (Hts) as proteins whose synaptic expression is restricted by miR-34. Manipulation of miR-34, Nrx-IV or Hts-M function in motor neurons or muscle supports a model where presynaptic miR-34 inhibits Nrx-IV to influence active zone formation, whereas, postsynaptic miR-34 inhibits Hts to regulate the initiation of bouton formation from presynaptic terminals.

Highlights

Micro(mi)RNA-based post-transcriptional regulatory mechanisms have been broadly implicated in the assembly and modulation of synaptic connections required to shape neural circuits, relatively few specific miRNAs have been identified that control synapse formation
Micro(mi)RNAs have emerged as ideal candidates to regulate nervous system development, plasticity and function1,2. miRNAs can concurrently regulate the stability and translation of distinct target mRNAs via complementary miRNA response elements (MREs) that rely on conserved “seed” sequences to confer robust yet imprecise base-pairing with target transcripts[3,4]
We found that ten of our novel miR-SP neuromuscular junction (NMJ) phenotypes match those of null alleles, including miR-13a, miR-14, miR-34, miR92a, miR-92b, miR-219, miR-277, miR-316, miR-973, and miR1014 (Fig. 1b compares mean miR-SP values in orange and null in blue for each confirmed hit normalized to their respective control strain)

Summary

Introduction

Micro(mi)RNA-based post-transcriptional regulatory mechanisms have been broadly implicated in the assembly and modulation of synaptic connections required to shape neural circuits, relatively few specific miRNAs have been identified that control synapse formation. After initial discovery of these small non-coding RNAs and key downstream loci by genetic analysis[5,6,7], genomic and informatics technologies were developed to identify many hundreds of conserved miRNAs and predict their direct target mRNAs in many animal species[8,9]. For this reason, we developed a collection of transgenic competitive inhibitors (“miRNA-SPonge” [miR-SP]) using the upstream activating sequence [UAS] control of the heterologous transcription factor GAL4 to provide spatio-temporal miRNA LOF in Drosophila[18,29]; this resource was made in a uniform genetic background ideal for quantitative phenotypic comparisons. Analysis of muscle form and function with the miR-SP collection revealed that endogenous levels of tissue-specific miRNA expression do not correlate well with functional impact[29], stressing the value of unbiased genetic screening to uncover novel functions

Methods

Results

Conclusion