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Glioblastoma (GBM) is a deadly brain tumor with a very poor prognosis. Development of new therapeutics is hindered by the lack of appropriate preclinical models that reflect the complexity of the tumor microenvironment, especially the crucial role of microglia. In this study, we investigated the impact of microglia on GBM models using humanized 3D spheroids. Homotypic and heterotypic spheroids were created out of a GBM-derived cell line (DKMG) or patient-derived glioma stem cells (GB22-13), along with a microglia cell line (HMC3). Heterotypic glioma-HMC3 spheroids exhibited increased proliferation and greater drug resistance to chemotherapy drug Temozolomide compared with homotypic spheroids. Heterotypic spheroids also grew larger, developed multinucleated structures within 7 days, and had a greater invasive potential. Additionally, a distinct core-shell structure emerged in the heterotypic spheroids, with glioma cells concentrated in the core and a surrounding layer of microglia forming a protective shell that appeared to hinder drug penetration to the tumor core. Further, heterotypic cells were able to induce migration and polarization of peripheral blood monocytes (THP-1) towards M2 phenotypes, increasing immune evasion. These findings highlight the critical role of microglia in GBM development and progression, demonstrating their contribution to both reduced drug diffusion and increased tumor growth.

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. Our results unveil a non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins. Here the authors describe that the human origin recognition complex subunit 1 (ORC1) binds to RNAs transcribed from genes with origins of replication at their TSS impacting origin activation.

During autophagy, the coordinated actions of autophagosomes and lysosomes result in the controlled removal of damaged intracellular organelles and superfluous substrates. The evolutionary conservation of this process and its requirement for maintaining cellular homeostasis emphasizes the need to better dissect the pathways governing its molecular regulation. In our previously performed high-content screen, we assessed the effect of 1530 RNA-binding proteins on autophagy. Among the top regulators, we identified the eukaryotic translation initiation factor 4A-3 (eIF4A3). Here we show that depletion of eIF4A3 leads to a potent increase in autophagosome and lysosome biogenesis and an enhanced autophagic flux. This is mediated by the key autophagy transcription factor, TFEB, which becomes dephosphorylated and translocates from the cytoplasm to the nucleus where it elicits an integrated transcriptional response. We further identified an exon-skipping event in the transcript encoding for the direct TFEB kinase, GSK3B, which leads to a reduction in GSK3B expression and activity. Through analysis of TCGA data, we found a significant upregulation of eIF4A3 expression across several cancer types and confirmed the potential relevance of this newly identified signaling axis in human tumors. Hence, our data suggest a previously unrecognized role for eIF4A3 as a gatekeeper of autophagy through the control of TFEB activation, revealing a new mechanism for autophagy regulation.

Amyotrophic Lateral Sclerosis (ALS) is a multisystemic neurodegenerative disorder, with accumulating evidence indicat- ing metabolic disruptions in the skeletal muscle preceding disease symptoms, rather than them manifesting as a secondary consequence of motor neuron (MN) degeneration. Hence, energy homeostasis is deeply implicated in the complex physi- opathology of ALS and skeletal muscle has emerged as a key therapeutic target. Here, we describe intrinsic abnormalities in ALS skeletal muscle, both in patient-derived muscle cells and in muscle cell lines with genetic knockdown of genes related to familial ALS, such as TARDBP (TDP-43) and FUS. We found a functional impairment of myogenesis that paral- lels defects of glucose oxidation in ALS muscle cells. We identified FOXO1 transcription factor as a key mediator of these metabolic and functional features in ALS muscle, via gene expression profiling and biochemical surveys in TDP-43 and FUS-silenced muscle progenitors. Strikingly, inhibition of FOXO1 mitigated the impaired myogenesis in both the genetically modified and the primary ALS myoblasts. In addition, specific in vivo conditional knockdown of TDP-43 or FUS orthologs (TBPH or caz) in Drosophila muscle precursor cells resulted in decreased innervation and profound dysfunction of motor nerve terminals and neuromuscular synapses, accompanied by motor abnormalities and reduced lifespan. Remarkably, these phenotypes were partially corrected by foxo inhibition, bolstering the potential pharmacological management of muscle intrinsic abnormalities associated with ALS. The findings demonstrate an intrinsic muscle dysfunction in ALS, which can be modulated by targeting FOXO factors, paving the way for novel therapeutic approaches that focus on the skeletal muscle as complementary target tissue.

Key Points The development of new methods for preparing and sequencing RNA sequencing libraries, as well as new alignment algorithms, have revealed many thousands of previously unknown non-canonical splicing events. Non-canonical splicing events are often tissue-specific and are particularly enriched in the central nervous system, thereby increasing proteome diversity or regulating gene expression. Cryptic exons, microexons and recursive splice sites often require unconventional exon definition mechanisms. Other non-canonical splicing events result from lower or higher splicing efficiency than normal (such as retained introns and exonic introns), changes in the usual order of splicing (circular RNAs and chimeric RNAs) or changes in the consensus sequence (atypical splice sites). Transposable elements are a rich source of newly emerging cryptic exons, which can contribute to the evolution of gene regulatory networks. Mutations that perturb functionally important non-canonical splicing events, or strongly increase the recognition of cryptic splice sites, can cause numerous diseases. Non-canonical splicing mechanisms offer new therapeutic opportunities to treat disease. Advances in transcriptomics and analysis have identified thousands of previously unknown non-canonical splicing events. In this Review, the authors discuss the mechanisms and functions of these events and their roles in a variety of diseases. They explain how non-canonical splicing mechanisms can be targeted or exploited for therapeutic strategies. Recent improvements in experimental and computational techniques that are used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimizes their potential to disrupt gene expression. We explain how non-canonical splicing can lead to aberrant transcripts that cause many diseases, and also how it can be exploited for new therapeutic strategies.

Objective SNUPN‐related muscular dystrophy or LGMDR29 is a new entity that covers from a congenital or childhood onset pure muscular dystrophy to more complex phenotypes combining neurodevelopmental features, cataracts, or spinocerebellar ataxia. So far, 12 different variants have been described. Here we report the first family with SNUPN‐related muscular dystrophy presenting an adult‐onset myopathy as well as novel ultrastructural findings. Methods Clinical evaluation, muscle and brain magnetic resonance imaging (MRI), and muscle histopathological and electron microscopy analysis were conducted. Functional studies including protein modelling and interaction, immunofluorescence and splicing analysis were also performed. Results Two siblings carrying two novel deleterious variants in the SNUPN gene (p.Arg27Cys and p.Cys174Tyr) showed adult‐onset proximo‐distal and axial muscle weakness with early respiratory involvement. One patient presented with asymptomatic cerebellar atrophy. Muscle MRI identified involvement in the paravertebral, triceps brachii, sartorius and gracilis muscles. The histopathology revealed dystrophic changes and an abnormal pattern of cytoskeletal and myofibrillar proteins, while electron microscopy disclosed the proliferation of granules and vesicles associated with features of nuclear envelope and sarcolemma remodelling. Functional studies showed that SNUPN variants impair snurportin‐1 function through reduced binding affinity to importin‐β and impaired folding, leading to disturbed nuclear import of small nuclear ribonucleoproteins and downstream splicing. Interpretation Our work expands the phenotype of SNUPN‐related muscular dystrophy and provides more insights into their pathological profile. We advise SNUPN testing in patients with late‐onset proximo‐distal and axial weakness with early respiratory impairment and features reminding inclusion body myositis (IBM). Granular deposits suggestive of biomolecular condensates perturbed cell organelle traffic and membrane homeostasis, opening new avenues to understand the pathomechanisms involved in this novel disease.

Highly conserved recursive splice sites are identified in vertebrates, particularly within long genes encoding proteins that are involved in neuronal development; analysis of the splicing mechanism reveals that such recursive splicing sites can be used to dictate different mRNA isoforms. Recursive splicing in insects and vertebrates The mechanisms by which the very longest genes in eukaryotic genomes are accurately processed are poorly understood. It was thought that intron removal generally involved a single excisive step. Later studies showed that, in flies, some introns contain internal splice sites that cause 'recursive splicing', in which single introns are removed 'bit-by-bit' in several sequential splicing reactions. Brenton Graveley and coworkers demonstrate that the scope of this regulatory mechanism is much more extensive in flies than had been appreciated. They identify nearly 200 zero-nucleotide exons in Drosophila that are the products of recursive splicing. Jernej Ule and colleagues identify recursive splicing sites in vertebrates, particularly within long genes encoding proteins that are involved in neuronal development. Analysis of the mechanism of their splicing reveals that such splicing sites can be used to dictate different mRNA isoforms. It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing 1 , 2 . The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an ‘RS-exon’ that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5′ splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5′ splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons.

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. We propose that fluctuating concentrations of RNA during the cell cycle may play a sequential role in controlling origins through interaction with this flexible region of ORC1. Our results unveil a novel non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins.Competing Interest StatementThe authors have declared no competing interest.

Liver kinase B1 (LKB1/STK11) is an important regulator of pancreatic β-cell identity and function. Elimination of from the β-cell results in improved glucose-stimulated insulin secretion and is accompanied by profound changes in gene expression, including the upregulation of several neuronal genes. The mechanisms through which LKB1 controls gene expression are, at present, poorly understood. Here, we explore the impact of β cell- selective deletion of on chromatin accessibility in mouse pancreatic islets. To characterize the role of LKB1 in the regulation of gene expression at the transcriptional level, we combine these data with a map of islet active transcription start sites and histone marks. We demonstrate that LKB1 elimination from β-cells results in widespread changes in chromatin accessibility, correlating with changes in transcript levels. Changes occurred in hundreds of promoter and enhancer regions, many of which were close to neuronal genes. We reveal that dysregulated enhancers are enriched in binding motifs for transcription factors important for β-cell identity, such as FOXA, MAFA or RFX6 and we identify microRNAs (miRNAs) that are regulated by LKB1 at the transcriptional level. Overall, our study provides important new insights into the epigenetic mechanisms by which LKB1 regulates β-cell identity and function.

Alternative splicing is an RNA processing used by the cell to increase its protein diversity and genome plasticity through generation of several transcripts from the same gene. It affects the vast majority of biological processes, from stem cell differentiation to cell metabolism. However, tools to properly study the role of a specific splice variant are still missing. With the discovery of the bacterial CRISPR system, a new era in nucleic acid editing has emerged. RNA-directed CRISPR/Cas13 RNAses were recently shown to efficiently target the RNA with higher specificity than Cas9 to the DNA. In this work, we are taking advantage of the catalytic dead mutant dCas13 family member dCasRx to edit alternative splicing patterns in a physiological context. Thanks to our new strategy, isoform-switching splicing changes are easily obtained at endogenous genes without impacting overall gene expression levels. Moreover, we propose a new application for this dCasRx splicing editing system to identify the key regulatory elements involved in the alternative splicing of a given gene. This new approach will increase the RNA toolkit to properly understand the biological impact and regulatory mechanisms of alternative splicing in a given biological process or pathological scenario. Competing Interest Statement The authors have declared no competing interest.