The long noncoding RNA nHOTAIRM1 is necessary for differentiation and activity of iPSC-derived spinal motor neurons

Abstract

The mammalian nervous system is made up of an extraordinary array of diverse cells that form intricate functional connections. The programs underlying cell lineage specification, identity and function of the neuronal subtypes are managed by regulatory proteins and RNAs, which coordinate the succession of steps in a stereotyped temporal order. In the central nervous system (CNS), motor neurons (MNs) are responsible for controlling essential functions such as movement, breathing, and swallowing by integrating signal transmission from the cortex, brainstem, and spinal cord (SC) towards peripheral muscles. A prime role in guiding the progression of progenitor cells towards the MN fate has been largely attributed to protein factors. More recently, the relevance of a class of regulatory RNAs abundantly expressed in the CNS - the long noncoding RNAs (lncRNAs) - has emerged overwhelmingly. LncRNA-driven gene expression control is key to regulating any step of MN differentiation and function, and its derangement profoundly impacts neuronal pathophysiology. Here, we uncover a novel function for the neuronal isoform of HOTAIRM1 (nHOTAIRM1), a lncRNA specifically expressed in the SC. Using a model system that recapitulates spinal MN (spMN) differentiation, we show that nHOTAIRM1 intervenes in the binary cell fate decision between MNs and interneurons, acting as a pro-MN factor. Furthermore, human iPSC-derived spMNs without nHOTAIRM1 display altered neurite outgrowth, with a significant reduction of both branch and junction numbers. Finally, the expression of genes essential for synaptic connectivity and neurotransmission is also profoundly impaired when nHOTAIRM1 is absent in spMNs. Mechanistically, nHOTAIRM1 establishes both direct and indirect interactions with a number of target genes in the cytoplasm, being a novel post-transcriptional regulator of MN biology. Overall, our results indicate that the lncRNA nHOTAIRM1 is essential for the specification of MN identity and the acquisition of proper morphology and synaptic activity of post-mitotic MNs.

Live-cell imaging of circular and long non-coding RNAs associated to FUS pathological aggregates by Pepper fluorescent RNA

“…To do that, we combined smFISH and immunofluorescence, targeting nHOTAIRM1, the SG marker G3BP1 and FUS. As previously reported, we observed that nHOTAIRM1 signal was distributed both in the nucleus and in the cytoplasm (Rea et al, 2020;Tollis et al, 2023) (Dormann et al, 2010;Kino et al, 2011). In FUS P525L MNs, where FUS de-localizes in the cytoplasm and participates in SG (Fig 4A, lower panel) (Bosco et al, 2010;Lenzi et al, 2015), nHOTAIRM1 was again recruited to SG.…”

“…Interestingly, the PCC between nHOTAIRM1 and DCP1A increased in FUS P525L conditions regardless of stress induction (Supplementary Fig 4B). As it was previously demonstrated that nHOTAIRM1 biochemically interacts with FUS (Rea et al, 2020;Tollis et al, 2023) and that mutant FUS binds and sequesters proteins involved in p-bodies formation (Takanashi and Yamaguchi, 2014), these data suggest that the presence of mutant FUS in the cytoplasm could drive the interaction between nHOTAIRM1 and p-bodies.…”

“…For this reason, we also decided to explore the behaviour of nHOTAIRM1, an attractive case study of lncRNA in an ALS-like context. The neuronal isoform of the lncRNA HOTAIRM1 (nHOTAIRM1) plays a crucial role in the control of neuronal differentiation (Rea et al, 2020; Tollis et al, 2023) and is highly expressed in MNs. nHOTAIRM1 has been shown to directly interact with the FUS protein in the cytoplasm (Rea et al, 2020), where FUS regulates its abundance.…”

LOC646762 Is Involved in Adipogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells

“…In recent years, functional genomics and transcriptome sequencing technologies have advanced rapidly, revealing an increasing number of factors involved in the regulation of adipogenic differentiation. Among these, long-chain noncoding RNAs (lncRNAs) have received increasing attention due to their important regulatory roles. , LncRNAs are the most abundant type of noncoding RNA and comprise >200 nt long transcripts that play important roles in cell differentiation, growth, development, and onset of many diseases. , LncRNAs exhibit multiple regulatory functions and participate in several important biological processes. They play important regulatory roles in the differentiation of MSCs into adipocytes; however, few studies have explored the role of lncRNAs in adipogenic differentiation .…”

“… 12 , 13 LncRNAs are the most abundant type of noncoding RNA and comprise >200 nt long transcripts that play important roles in cell differentiation, growth, development, and onset of many diseases. 14 , 15 LncRNAs exhibit multiple regulatory functions and participate in several important biological processes. They play important regulatory roles in the differentiation of MSCs into adipocytes; however, few studies have explored the role of lncRNAs in adipogenic differentiation.…”