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5-ethynyluridine perturbs nuclear RNA metabolism to promote the nuclear accumulation of TDP-43 and other RNA binding proteins

ABSTRACT: TDP-43, an essential nucleic acid binding protein and splicing regulator, is disrupted in neurodegeneration and an urgent therapeutic target. We and others demonstrated that TDP-43 nuclear localization and function depend on the abundance of its nuclear pre-mRNA binding partners and its recruitment into large ribonucleoprotein complexes, which restricts TDP-43 nuclear efflux and maintains the critical nuclear pool of TDP-43. To further investigate the interplay between TDP-43 and nascent RNAs, we set out to employ 5-ethynyluridine (5EU), a widely used uridine analog that enables ‘click chemistry’ labeling of nascent RNAs. Unexpectedly, 5EU induced the nuclear accumulation of TDP-43 and other nuclear RNA binding proteins (RBPs) and attenuated TDP-43 nuclear exit. RNA FISH demonstrated 5EU-induced nuclear accumulation of polyadenylated and GU-rich RNAs. TDP-43 eCLIP confirmed that 5EU stabilizes TDP-43 binding to intronic sites, and TDP-43 function in cryptic exon repression remained intact. There was no major change in RNA stability, but RNA-seq demonstrated a marked alteration in RNA splicing, which is less efficient in 5EU-treated cells. Thus, 5EU perturbs nuclear RNA metabolism leading to nuclear RNA and RBP accumulation. These findings have critical implications for studies utilizing 5EU and identify a novel mechanistic inroad to support TDP-43 homeostasis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE292953 | GEO | 2026/03/25

REPOSITORIES: GEO

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