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The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro -derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-associated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUS P525L mutation associated with ALS. The RNA binding protein FUS functions in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). Here the authors show that FUS controls back-splicing reactions leading to circular RNA (circRNA) production in stem cell-derived motor neurons and that ALS-associated FUS mutations affect the biogenesis of circRNAs.

Neuronal differentiation is a timely and spatially regulated process, relying on precisely orchestrated gene expression control. The sequential activation/repression of genes driving cell fate specification is achieved by complex regulatory networks, where transcription factors and noncoding RNAs work in a coordinated manner. Herein, we identify the long noncoding RNA HOTAIRM1 (HOXA Transcript Antisense RNA, Myeloid-Specific 1) as a new player in neuronal differentiation. We demonstrate that the neuronal-enriched HOTAIRM1 isoform epigenetically controls the expression of the proneural transcription factor NEUROGENIN 2 that is key to neuronal fate commitment and critical for brain development. We also show that HOTAIRM1 activity impacts on NEUROGENIN 2 downstream regulatory cascade, thus contributing to the achievement of proper neuronal differentiation timing. Finally, we identify the RNA-binding proteins HNRNPK and FUS as regulators of HOTAIRM1 biogenesis and metabolism. Our findings uncover a new regulatory layer underlying NEUROGENIN 2 transitory expression in neuronal differentiation and reveal a previously unidentified function for the neuronal-induced long noncoding RNA HOTAIRM1.

Medulloblastoma (MB) is the most common pediatric brain tumor and a primary cause of cancer-related death in children. Until a few years ago, only clinical and histological features were exploited for MB pathological classification and outcome prognosis. In the past decade, the advancement of high-throughput molecular analyses that integrate genetic, epigenetic, and expression data, together with the availability of increasing wealth of patient samples, revealed the existence of four molecularly distinct MB subgroups. Their further classification into 12 subtypes not only reduced the well-characterized intertumoral heterogeneity, but also provided new opportunities for the design of targets for precision oncology. Moreover, the identification of tumorigenic and self-renewing subpopulations of cancer stem cells in MB has increased our knowledge of its biology. Despite these advancements, the origin of MB is still debated, and its molecular bases are poorly characterized. A major goal in the field is to identify the key genes that drive tumor growth and the mechanisms through which they are able to promote tumorigenesis. So far, only protein-coding genes acting as oncogenic drivers have been characterized in each MB subgroup. The contribution of the non-coding side of the genome, which produces a plethora of transcripts that control fundamental biological processes, as the cell choice between proliferation and differentiation, is still unappreciated. This review wants to fill this major gap by summarizing the recent findings on the impact of non-coding RNAs in MB initiation and progression. Furthermore, their potential role as specific MB biomarkers and novel therapeutic targets is also highlighted.

Mutations in fused in sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS). FUS is a multifunctional protein involved in the biogenesis and activity of several types of RNAs, and its role in the pathogenesis of ALS may involve both direct effects of disease-associated mutations through gain- and loss-of-function mechanisms and indirect effects due to the cross talk between different classes of FUS-dependent RNAs. To explore how FUS mutations impinge on motor neuron-specific RNA-based circuitries, we performed transcriptome profiling of small and long RNAs of motor neurons (MNs) derived from mouse embryonic stem cells carrying a FUS-P517L knock-in mutation, which is equivalent to human FUS-P525L, associated with a severe and juvenile-onset form of ALS. Combining ontological, predictive and molecular analyses, we found an inverse correlation between several classes of deregulated miRNAs and their corresponding mRNA targets in both homozygous and heterozygous P517L MNs. We validated a circuitry in which the upregulation of miR-409-3p and miR-495-3p, belonging to a brain-specific miRNA subcluster implicated in several neurodevelopmental disorders, produced the downregulation of Gria2, a subunit of the glutamate α‐amino‐3‐hydroxy‐5‐methyl-4-isoxazole propionic acid (AMPA) receptor with a significant role in excitatory neurotransmission. Moreover, we found that FUS was involved in mediating such miRNA repression. Gria2 alteration has been proposed to be implicated in MN degeneration, through disturbance of Ca 2+ homeostasis, which triggers a cascade of damaging “excitotoxic” events. The molecular cross talk identified highlights a role for FUS in excitotoxicity and in miRNA-dependent regulation of Gria2. This circuitry also proved to be deregulated in heterozygosity, which matches the human condition perfectly.

The transcription factor ID2 is an important repressor of neural differentiation strongly implicated in nervous system cancers. MicroRNAs (miRNAs) are increasingly involved in differentiation control and cancer development. Here we show that two miRNAs upregulated on differentiation of neuroblastoma cells--miR-9 and miR-103--restrain ID2 expression by directly targeting the coding sequence and 3' untranslated region of the ID2 encoding messenger RNA, respectively. Notably, the two miRNAs show an inverse correlation with ID2 during neuroblastoma cell differentiation induced by retinoic acid. Overexpression of miR-9 and miR-103 in neuroblastoma cells reduces proliferation and promotes differentiation, as it was shown to occur upon ID2 inhibition. Conversely, an ID2 mutant that cannot be targeted by either miRNA prevents retinoic acid-induced differentiation more efficient than wild-type ID2. These findings reveal a new regulatory module involving two microRNAs upregulated during neural differentiation that directly target expression of the key differentiation inhibitor ID2, suggesting that its alteration may be involved in neural cancer development.

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.

MicroRNAs (miRNAs) are tiny noncoding RNAs whose function as modulators of gene expression is crucial for the proper control of cell growth and differentiation. Although the profile of miRNA expression has been defined for many different cellular systems, the elucidation of the regulatory networks in which they are involved is only just emerging. In this work, we identify a crucial role for three neuronal miRNAs (9, 125a, and 125b) in controlling human neuroblastoma cell proliferation. We show that these molecules act in an additive manner by repressing a common target, the truncated isoform of the neurotrophin receptor tropomyosin-related kinase C, and we demonstrate that the down-regulation of this isoform is critical for regulating neuroblastoma cell growth. Consistently with their function, these miRNAs were found to be down-modulated in primary neuroblastoma tumors.

Glioblastomas (GBM) are the most aggressive form of primary brain tumors in humans. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population of defined Glioblastoma Stem cells (GSCs) was reported. Increased expression of anti-apoptotic and chemo-resistance genes in GCSs subpopulation favors their high resistance to a broad spectrum of drugs. Our previous studies showed the ability of M2 muscarinic receptors to negatively modulate the cell growth in GBM cell lines and in the GSCs. The aim of this study was to better characterize the inhibitory effects of M2 receptors on cell proliferation and survival in GSCs and investigate the molecular mechanisms underlying the M2-mediated cell proliferation arrest and decreased survival. Moreover, we also evaluated the ability of M2 receptors to interfere with Notch1 and EGFR pathways, whose activation promotes GSCs proliferation. Our data demonstrate that M2 receptors activation impairs cell cycle progression and survival in the primary GSC lines analyzed (GB7 and GB8). Moreover, we also demonstrated the ability of M2 receptor to inhibit Notch1 and EGFR expression, highlighting a molecular interaction between M2 receptor and the Notch-1/EGFR pathways also in GSCs.

RNA metabolism is central to cellular physiopathology. Almost all the molecular pathways underpinning biological processes are affected by the events governing the RNA life cycle, ranging from transcription to degradation. The deregulation of these processes contributes to the onset and progression of human diseases. In recent decades, considerable efforts have been devoted to the characterization of noncoding RNAs (ncRNAs) and to the study of their role in the homeostasis of the nervous system (NS), where they are highly enriched. Acting as major regulators of gene expression, ncRNAs orchestrate all the steps of the differentiation programs, participate in the mechanisms underlying neural functions, and are crucially implicated in the development of neuronal pathologies, among which are neurodegenerative diseases. This review aims to explore the link between ncRNA dysregulation and amyotrophic lateral sclerosis (ALS), the most frequent motoneuron (MN) disorder in adults. Notably, defective RNA metabolism is known to be largely associated with this pathology, which is often regarded as an RNA disease. We also discuss the potential role that these transcripts may play as diagnostic biomarkers and therapeutic targets.

Small nucleolar RNAs (snoRNAs) play a key role in eukaryotic ribosome biogenesis. In most cases, snoRNAs are encoded in introns and are released through the splicing reaction. Some snoRNAs are, instead, produced by an alternative pathway consisting of endonucleolytic processing of pre-mRNA. XendoU, the endoribonuclease responsible for this activity, is a U-specific, metaldependent enzyme that releases products with 2'-3' cyclic phosphate termini. XendoU is broadly conserved among eukaryotes, and it is a genetic marker of nidoviruses, including the severe acute respiratory syndrome coronavirus, where it is essential for replication and transcription. We have determined by crystallography the structure of XendoU that, by refined search methodologies, appears to display a unique fold. Based on sequence conservation, mutagenesis, and docking simulations, we have identified the active site. The conserved structural determinants of this site may provide a framework for attempting to design antiviral drugs to interfere with the infectious nidovirus life cycle.