Objective: This study aims to explore the possible pathogenic mechanisms of FCMTE1 from the perspectives of RNA toxicity and RNA binding protein.

Background: FCMTE1 is characterized by intronic (TTTCA)exp or (TTTGA)exp expansions leading to RNA toxicity. While RNA toxicity has been implicated in FCMTE1 pathogenesis, the mechanisms remain unclear. NOVA2 is a neuron-specific RNA-binding protein that regulates alternative splicing of genes essential for neuronal function. Disruption of NOVA2 function has been associated with neurological disorders.

Method: Nuclear proteins from SH-SY5Y cells and mouse brains were incubated with biotinylated repeat RNAs for streptavidin pulldown assays. Binding specificity was validated via western blot, EMSA, and immunofluorescence in N2A cells. RNA-seq and rMATS analysis of FCMTE1 patient iPSC-derived neurons identified splicing alterations, which were cross-referenced with Nova1/2 knockout (KO) mouse datasets.

Results: 13 proteins were captured by (UUUUA)₃₅(UUUCA)₁₁₀ RNA and 14 proteins were captured by (UUUUA)₁₈(UUUGA)₇₃ RNA in both nuclear SH-SH5Y and mouse whole brain. Western blot analysis confirmed the preferential binding of NOVA2, HNRPL and HNRLL to biotinylated (UUUUA)₃₅(UUUCA)₁₁₀ RNA in SH-SY5Y nuclear extract, compared to (UUUUA)₁₆ and (UUUUA)₁₈(UUUGA)₇₃ RNA. Immunofluorescence revealed that (UUUCA)exp RNA aggregates disrupted the nuclear distribution of NOVA2 in Neuro-2a cells. EMSA confirmed direct, length-dependent binding of NOVA2 to (UUUCA)exp RNA. RNA sequencing of FCMTE1 iPSC-derived neurons identified 927 differentially spliced genes, with significant enrichment of NOVA binding motifs in skipped exons. Notably, 46 alternatively spliced genes overlapped with known NOVA targets, including synaptic genes such as DLG3, ADAM22, and SNAP25.

Conclusion: Our study provides the first evidence that (UUUCA)exp RNA aggregates disrupt nuclear organization of NOVA2, impairing splicing fidelity in synaptic genes. This bridges RNA toxicity to synaptic instability and excitatory-inhibitory imbalance, providing the first evidence of splicing dysregulation in FCMTE1. These results uncover a new facet of FCMTE1 pathogenesis and highlight the potential for targeting splicing regulatory pathways in therapeutic strategies.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/disrupted-nova2-mediated-splicing-by-tttca-repeat-expansion-unveiling-aberrant-synaptic-gene-regulation-in-fcmte1/