A multifunctional locus controls motor neuron differentiation through short and long non coding RNAs

The transition from dividing progenitors into post-mitotic motor neurons is orchestrated by a series of events mainly studied at the transcriptional level by analyzing the activity of specific programming transcription factors. In this work, we describe that a motor neuron-specific transcriptional unit, harboring a lncRNA (lncMN2-203) and two miRNAs (miR-325-3p and miR-384-5p), controls such transition at the post-transcriptional level. Through the use of in vitro differentiation of mESC and single-cell sequencing of CRISPR/Cas9 mutants, we demonstrate that lncMN2-203 affects motor neuron differentiation by sponging miR-466i-5p and up-regulating its targets; among them Onecut2, which is the upstream regulator of the MN-specific programming factor Islet-1. In synergy with lncMN2-203, instead the depletion of the co-transcribed miR-325-3p and miR-384-5p was found to mainly sustain proliferation-related factors. These findings indicate the functional relevance of the MN2 locus and contribute to elucidate additional layers of regulation controlling the specificity of motor neuron differentiation. Overall design: CRISPR/Cas9 gene editing approach was applied to generate loss-of-function mutant mESCs of MN2 locus (DeltaSP and KO). Control (WT) and mutants cells were differentiated to Embroyd Bodies at day6 (EB6) according to MN differentiation protocol published in Wichterle and Peljto, 2008. For each condition two clones were analyzed with single cell RNA sequencing (10X Genomics Chromium, Chromium Single Cell 3' Solution V3). FASTQ reads were aligned, filtered and counted through the Cell Ranger pipeline (v4.0) using standard parameters. The Mm10 genome version was used in alignment step and annotation refers to Ensembl Release93. See methods for more details.