Abstract
Although long non-coding RNAs (lncRNAs) is one of the most abundant classes of RNAs encoded within the mammalian genome and are highly expressed in the adult brain, they remain poorly characterized and their roles in the brain development are not well understood. Here we identify the lncRNA Lacuna (also catalogued as NONMMUT071331.2 in NONCODE database) as a negative regulator of neuronal differentiation in the neural stem/progenitor cells (NSCs) during mouse brain development. In particular, we show that Lacuna is transcribed from a genomic locus near to the Tbr2/Eomes gene, a key player in the transition of intermediate progenitor cells towards the induction of neuronal differentiation. Lacuna RNA expression peaks at the developmental time window between E14.5 and E16.5, consistent with a role in neural differentiation. Overexpression experiments in ex vivo cultured NSCs from murine cortex suggest that Lacuna is sufficient to inhibit neuronal differentiation, induce the number of Nestin+ and Olig2+ cells, without affecting proliferation or apoptosis of NSCs. CRISPR/dCas9-KRAB mediated knockdown of Lacuna gene expression leads to the opposite phenotype by inducing neuronal differentiation and suppressing Nestin+ and Olig2+ cells, again without any effect on proliferation or apoptosis of NSCs. Interestingly, despite the negative action of Lacuna on neurogenesis, its knockdown inhibits Eomes transcription, implying a simultaneous, but opposite, role in facilitating the Eomes gene expression. Collectively, our observations indicate a critical function of Lacuna in the gene regulation networks that fine tune the neuronal differentiation in the mammalian NSCs.
Highlights
Understanding the molecular mechanisms that control the mammalian brain development is one of the most challenging goals of biomedical sciences
We and others had previously reported that a number of long non-coding RNAs (lncRNAs) genes are found in close genomic proximity to genes encoding for transcription factors with critical regulatory roles in neural development (Antoniou et al, 2014; Ponjavic et al, 2009)
The complexity of the mammalian brain is mainly due to the huge numbers of neurons and glial cells that interact to form its underlying structure. All these cells are derived from a pool of neural stem cells that proliferate with enormous rates and differentiate to generate first neurons and glial cells
Summary
Understanding the molecular mechanisms that control the mammalian brain development is one of the most challenging goals of biomedical sciences. Long non-coding RNAs (lncRNAs) are transcripts larger than 200 nt that can be modified by 5′-capping, polyadenylation and splicing, similar to mRNAs, yet they are not translated into proteins (Maeda et al, 2006; Djebali et al, 2012) Their genomic location varies as they can be found in introns of protein coding genes, sense or antisense to other genes, intergenic regions (Kapranov et al, 2007; Seila et al, 2008), promoters (Hung et al, 2011), enhancers (Ørom et al, 2010), gene regulatory regions like UTRs (Mercer et al, 2011), even telomeres (Azzalin et al, 2007). LncRNAs appear to be involved in the regulatory networks that control stem cell pluripotency, carcinogenesis, growth, and function of many tissues and organs (Mercer et al, 2009; Sheik Mohamed et al, 2010; Guttman et al, 2011; Ng et al, 2012; Antoniou et al, 2014; Ramos et al, 2015; Giakountis et al, 2016; Zarkou et al, 2018; Chi et al, 2019; Malissovas et al, 2019)
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