RNA-protein interactions play a key role in the aberrant splicing of CFTR exon 9. Exon 9 skipping leads to the production of a nonfunctional chloride channel associated with severe forms of cystic fibrosis. The missplicing depends on TDP-43 binding to an extended UG-rich binding site upstream of CFTR exon 9 3' splicing site (3'ss) and is associated with concomitant hnRNP A1 recruitment. Although TDP-43 is the dominant inhibitor of exon 9 inclusion, the role of hnRNP A1, a protein with two RNA recognition motifs, remained unclear. In this work, we have studied the interaction between hnRNP A1 and the CFTR pre-mRNA using NMR spectroscopy and Isothermal Titration Calorimetry. The affinities are submicromolar, and Isothermal Titration Calorimetry data suggest complexes with a 1:1 stoichiometry. NMR titrations reveal that hnRNP A1 interacts with model CTFR 3'ss sequences in a fast exchange regime at the NMR timescale. Splicing assays finally show that this hnRNP A1 binding site represents a previously unknown exonic splicing silencer element. Together, our results shed light on the mechanism of aberrant CFTR exon 9 splicing.

• MeSH
• Alternative Splicing genetics   MeSH
• Cystic Fibrosis genetics   MeSH
• Exons genetics   MeSH
• Heterogeneous Nuclear Ribonucleoprotein A1 * metabolism   genetics   MeSH
• Humans MeSH
• RNA Splice Sites genetics   MeSH
• RNA Precursors genetics   metabolism   MeSH
• Cystic Fibrosis Transmembrane Conductance Regulator * genetics   metabolism   MeSH
• RNA Splicing * genetics   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• CFTR protein, human MeSH Browser
• Heterogeneous Nuclear Ribonucleoprotein A1 * MeSH
• hnRNPA1 protein, human MeSH Browser
• RNA Splice Sites MeSH
• RNA Precursors MeSH
• Cystic Fibrosis Transmembrane Conductance Regulator * MeSH

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1, which involve human-specific mechanisms2-5 that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors6. Single-cell transcriptomics and comparison to independent neural stem cells7 showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids8. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.

• MeSH
• Amyotrophic Lateral Sclerosis * metabolism   pathology   MeSH
• C-Reactive Protein * metabolism   MeSH
• DNA-Binding Proteins * deficiency   metabolism   MeSH
• Frontotemporal Lobar Degeneration * metabolism   pathology   MeSH
• Humans MeSH
• Nerve Net * metabolism   pathology   MeSH
• Neural Stem Cells cytology   MeSH
• Neuroglia cytology   MeSH
• Neurons * cytology   metabolism   MeSH
• Nerve Tissue Proteins * metabolism   MeSH
• Reproducibility of Results MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• C-Reactive Protein * MeSH
• DNA-Binding Proteins * MeSH
• neuronal pentraxin MeSH Browser
• Nerve Tissue Proteins * MeSH
• TARDBP protein, human MeSH Browser