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

Abstract

The transition from dividing progenitors to postmitotic motor neurons (MNs) is orchestrated by a series of events, which are mainly studied at the transcriptional level by analyzing the activity of specific programming transcription factors. Here, we identify a posttranscriptional role of a MN-specific transcriptional unit (MN2) harboring a lncRNA (lncMN2-203) and two miRNAs (miR-325-3p and miR-384-5p) in this transition. Through the use of in vitro mESC differentiation and single-cell sequencing of CRISPR/Cas9 mutants, we demonstrate that lncMN2-203 affects MN differentiation by sponging miR-466i-5p and upregulating its targets, including several factors involved in neuronal differentiation and function. In parallel, miR-325-3p and miR-384-5p, co-transcribed with lncMN2-203, act by repressing proliferation-related factors. These findings indicate the functional relevance of the MN2 locus and exemplify additional layers of specificity regulation in MN differentiation.

Advances in endogenous RNA pull-down: A straightforward dextran sulfate-based method enhancing RNA recovery

“…As an example, we tested the performance of the new set-up on lncMN2-203, whose isolation from 1 mg of DSS-treated extracts was found enhanced. Moreover, we found that the DSS treatment also improved the recovery of miR-466i-5p, previously identified as a functional lncMN2-203 partner (Carvelli et al, 2022). In line with the binding specificity, only background enrichments were found for miR-669a-5p and miR-325-3p, previously shown as non-interacting miRNAs.…”

“…In this setting, we also checked for miRNAs known to co-precipitate with lncMN2-203 and we confirmed that miR-466i-5p was significantly enriched in the MN2 as compared to the U1 PD (Figure 1C, left panel). MiR-669a-5p and miR-325-3p, used as negative controls, were almost undetectable in both the conditions (Figure 1C, middle and right panels), in line with their known inability to interact with the lncRNA (Carvelli et al, 2022).…”

“…The efficacy of the procedure was tested on lncMN2-203 ( Figure 1A ), a motor neuron (MN) specific lncRNA ( Biscarini et al, 2018 ) recently found to control the specification of murine MNs by acting as a miRNA sponge ( Carvelli et al, 2022 ). By using the same amount of total cell extract (2 mg of Input) previously used for lncMN2-203 precipitation ( Carvelli et al, 2022 ), we reproduced its significant enrichment as compared to U1 snRNA PD, used as a control ( Figure 1B ). In accordance with the earlier analyses, the recovery of the other lncRNA isoforms (lncMN2-202/204) was almost null, in both the specific and control samples.…”

“…Hereinafter, we propose a strategy which represents a variation on the theme of the standard RNA pull-down (PD), with the innovative use of the Dextran Sulphate Sodium (DSS) salt as a hygroscopic chemical additive. By testing the procedure to the motoneuron-expressed lncRNA lncMN2-203 ( Biscarini et al, 2018 ; Carvelli et al, 2022 ), we found that DSS 1) greatly improves the purification of lncMN2-203, as compared to previous analysis and, importantly, 2) facilitates the identification of its RNA binding partner, the microRNA miRNA-466i-5p. These results promise to upgrade RNA functional analyses in a straightforward and effective manner.…”

“…FIGURE 1LncMN2-203 RNA pull-down in mESCs-derived embryoid bodies. (A) Block-and-line scheme representing the structure of the three lncMN2 RNA isoforms(Carvelli et al, 2022). Exon (black blocks)/intron (thin lines).…”