Synergism between microRNA-124 and ELAVL3 drives neuronal gene upregulation during direct neuronal reprogramming of human fibroblasts
2020
Human neurons generated by direct conversion of adult fibroblasts can serve instrumental tools for modeling diseases as well as a platform to study neurogenic processes. Neuron-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), direct cell fate switching of human fibroblasts to neurons when ectopically expressed. A critical component of the miRNA-mediated conversion process is the role of miR-9/9*-124 targeting and repressing anti-neurogenic genes. However, how these miRNAs function beyond the onset of the neuronal program remains unclear. Here, we performed the Argonaute (AGO) HITS-CLIP analysis to map direct target transcripts of miR-9/9*-124 when cells start adopting the neuronal fate during the conversion (reprogramming) process. Surprisingly, we found that miR-124, in particular, targets a suite of genes associated with neuronal differentiation and function, and these targets appeared to be upregulated by miR-124, as knocking down miR-124 function diminished the expression of these targets. A critical example of the upregulated miR-124 target genes is PTBP2, a neuron-enriched RNA-binding protein known to be repressed by its homolog PTBP1. We elected to focus on PTBP2 to gain insights into the mechanism underlying the activity of miR-124 as an inducer of its target genes. Paradoxically, both PTBP1 and PTBP2 are repressive targets of miR-124 in non-neuronal cells, yet PTBP2 selectively escapes the repression and becomes enriched in neurons. By interrogating 3′UTR sequences of PTBP1 and PTBP2, we provide evidence that the switching of miR-124 activity to an activator is determined by the sequence availability at the target transcript that allows the binding of neuronal RNA-binding proteins, in particular ELAVL3 for select upregulation of PTBP2. ELAVL3 binds and interacts with AGO at PTBP2 3′UTR, yet ELAVL3 binding alone without miR-124 sites is insufficient to trigger the upregulation of PTBP2. Knocking down ELAVL3 expression during neuronal reprogramming leads to PTBP2 downregulation and impairs neuronal conversion, highlighting the concerted interplay between miR-124 and ELAVL3 that drives neuronal fate acquisition. By elucidating how miR-124 promotes PTBP2 induction, our study lends support to the underexplored role of miRNAs in promoting target gene expression in neurons.
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