A direct regulatory link between microRNA-137 and SHANK2: implications for neuropsychiatric disorders

Background: Mutations in the SHANK genes, which encode postsynaptic scaffolding proteins, have been linked to a spectrum of neurodevelopmental disorders. The SHANK genes and the schizophrenia-associated microRNA-137 show convergence on several levels, as they are both expressed at the synapse, influence neuronal development, and have a strong link to neurodevelopmental and neuropsychiatric disorders like intellectual disability, autism, and schizophrenia. This compiled evidence raised the question if the SHANKs might be targets of miR-137. Methods: In silico analysis revealed a putative binding site for microRNA-137 (miR-137) in the SHANK2 3′UTR, while this was not the case for SHANK1 and SHANK3. Luciferase reporter assays were performed by overexpressing wild type and mutated SHANK2-3′UTR and miR-137 in human neuroblastoma cells and mouse primary hippocampal neurons. miR-137 was also overexpressed or inhibited in hippocampal neurons, and Shank2 expression was analyzed by quantitative real-time PCR and Western blot. Additionally, expression levels of experimentally validated miR-137 target genes were analyzed in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia and control individuals using the RNA-Seq data from the CommonMind Consortium. Results: miR-137 directly targets the 3′UTR of SHANK2 in a site-specific manner. Overexpression of miR-137 in mouse primary hippocampal neurons significantly lowered endogenous Shank2 protein levels without detectable influence on mRNA levels. Conversely, miR-137 inhibition increased Shank2 protein expression, indicating that miR-137 regulates SHANK2 expression by repressing protein translation rather than inducing mRNA degradation. To find out if the miR-137 signaling network is altered in schizophrenia, we compared miR-137 precursor and miR-137 target gene expression in the DLPFC of schizophrenia and control individuals using the CommonMind Consortium RNA sequencing data. Differential expression of 23% (16/69) of known miR-137 target genes was detected in the DLPFC of schizophrenia individuals compared with controls. We propose that in further targets (e.g., SHANK2, as described in this paper) which are not regulated on RNA level, effects may only be detectable on protein level. Conclusion: Our study provides evidence that a direct regulatory link exists between miR-137 and SHANK2 and supports the finding that miR-137 signaling might be altered in schizophrenia

Additional file 1: of A direct regulatory link between microRNA-137 and SHANK2: implications for neuropsychiatric disorders

Figure S1. Images of primary neuronal cultures pre‐ and posttreatment. Figure S2. High conservation of the miR‐137 binding site in the SHANK2‐3’UTR and of miR‐137 between different species. Figure S3. Relative expression levels of miR‐137 in different human tissues. Figure S4. Uncropped Western blot pictures. Table S1. ASD risk genes which are predicted or validated miR‐137 targets. Table S2. Primers used for cloning, mutagenesis, and screening. Primer sequences are all shown in 5′→3′ orientation. Table S3. Origin of total RNA samples used to measure hsa‐miR‐137 relative expression across different tissues (see Additional file 1: Figure S2 for results). Table S4. Experimentally validated miR‐137 targets. Table S5. Gene expression analysis of 69 validated miR‐137 target genes (including SHANK2) in the CommonMind RNA sequencing data. Table S6a. Gene expression analysis of validated targets from five different control microRNAs in the CommonMind RNA sequencing data. Genes labeled in gray withstand correction for multiple testing using the Benjamini-Hochberg method and a FDR of 10%. Table S6b. Comparison of the number of differentially expressed target genes of different microRNAs between SCZ and control individuals in the CommonMind RNASeq data. Table S7. Analysis of the 3′UTR of the differentially expressed miR-137 genes in the DLPFC between SCZ and control individuals for additional putative miR-124 and miR-128 binding sites. (PDF 1417 kb