Autism-associated miR-873 regulates ARID1B, SHANK3 and NRXN2 involved in neurodevelopment

Jing Lu,Helena C. Parkington,Sarah Williams,Mary A. Tonta,Yan Zhu,Michelle Watts,Charles Claudianos,Harold A. Coleman

doi:10.1038/s41398-020-01106-8

Abstract

Autism spectrum disorders (ASD) are highly heritable neurodevelopmental disorders with significant genetic heterogeneity. Noncoding microRNAs (miRNAs) are recognised as playing key roles in development of ASD albeit the function of these regulatory genes remains unclear. We previously conducted whole-exome sequencing of Australian families with ASD and identified four novel single nucleotide variations in mature miRNA sequences. A pull-down transcriptome analysis using transfected SH-SY5Y cells proposed a mechanistic model to examine changes in binding affinity associated with a unique mutation found in the conserved ‘seed’ region of miR-873-5p (rs777143952: T > A). Results suggested several ASD-risk genes were differentially targeted by wild-type and mutant miR-873 variants. In the current study, a dual-luciferase reporter assay confirmed miR-873 variants have a 20-30% inhibition/dysregulation effect on candidate autism risk genes ARID1B, SHANK3 and NRXN2 and also confirmed the affected expression with qPCR. In vitro mouse hippocampal neurons transfected with mutant miR-873 showed less morphological complexity and enhanced sodium currents and excitatory neurotransmission compared to cells transfected with wild-type miR-873. A second in vitro study showed CRISPR/Cas9 miR-873 disrupted SH-SY5Y neuroblastoma cells acquired a neuronal-like morphology and increased expression of ASD important genes ARID1B, SHANK3, ADNP2, ANK2 and CHD8. These results represent the first functional evidence that miR-873 regulates key neural genes involved in development and cell differentiation.

Highlights

Autism spectrum disorders (ASD) are characterised by impaired social interaction, communication deficits, restricted and repetitive behaviour, and affect 1–2% of the world population
miRNA regulatory elements (MREs) were predicted with MIRANDA32, ~200–300 bp gene sequence containing MRE was cloned into ΨCheck-2 vector before Renilla luciferase
Identification of mRNA targets of WT and Mut miRNA-873 Our previously reported pull-down transcriptome analysis used biotin-conjugated miRNA mimics to identify bound mRNA targets in human neuroblastoma SH-SY5Y cells and results were deposited in GenBank[16]

Summary

Introduction

Autism spectrum disorders (ASD) are characterised by impaired social interaction, communication deficits, restricted and repetitive behaviour, and affect 1–2% of the world population. Extensive research has been conducted to identify the genetic causes of ASD with literally hundreds of genes linked to ASD risk[1]. There are 913 genes represented in the Human Gene Module from Simons Foundation Autism Research Initiative (SFARI), a comprehensive resource on ASD genetics[2]. Significant efforts have been made to examine the role of DNA variations found in protein coding genes, there remains a paucity of knowledge concerning the large number of variations detected in noncoding DNA including microRNAs (miRNAs) and their target sites in mRNAs5. RNAs that regulate gene expression at all known levels of cell development, including the nervous system[6,7,8,9], and are thought to play a vital role in autism[10,11,12].

Methods

Results

Conclusion