Multivalent GU-rich oligonucleotides sequester TDP-43 in the nucleus by inducing high molecular weight RNP complexes.

TDP-43 nuclear clearance and cytoplasmic aggregation are hallmarks of TDP-43 proteinopathies. We recently demonstrated that binding to endogenous nuclear GU-rich RNAs sequesters TDP-43 in the nucleus by restricting its passive nuclear export. Here, we tested the feasibility of synthetic RNA oligonucleotide-mediated augmentation of TDP-43 nuclear localization. Using biochemical assays, we compared the ability of GU-rich oligonucleotides to engage in multivalent, RRM-dependent binding with TDP-43. When transfected into cells, (GU)16 attenuated TDP-43 mislocalization induced by transcriptional blockade or RanGAP1 ablation. Clip34nt and (GU)16 accelerated TDP-43 nuclear re-import after cytoplasmic mislocalization. RNA pulldowns confirmed that multivalent GU-oligonucleotides induced high molecular weight RNP complexes, incorporating TDP-43 and possibly other GU-binding proteins. Transfected GU-repeat oligos disrupted TDP-43 cryptic exon repression, likely by diverting TDP-43 from endogenous RNAs, except for Clip34nt that contains interspersed A and C. Thus, exogenous multivalent GU-RNAs can promote TDP-43 nuclear localization, though pure GU-repeat motifs impair TDP-43 function.

Elevated nuclear TDP-43 induces constitutive exon skipping.

BACKGROUND: Cytoplasmic inclusions and loss of nuclear TDP-43 are key pathological features found in several neurodegenerative disorders, suggesting both gain- and loss-of-function mechanisms of disease. To study gain-of-function, TDP-43 overexpression has been used to generate in vitro and in vivo model systems. METHODS: We analyzed RNA-seq datasets from mouse and human neurons overexpressing TDP-43 to explore species specific splicing patterns. We explored the dynamics between TDP-43 levels and exon repression in vitro. Furthermore we analyzed human brain samples and publicly available RNA datasets to explore the relationship between exon repression and disease. RESULTS: Our study shows that excessive levels of nuclear TDP-43 protein lead to constitutive exon skipping that is largely species-specific. Furthermore, while aberrant exon skipping is detected in some human brains, it is not correlated with disease, unlike the incorporation of cryptic exons that occurs after loss of TDP-43. CONCLUSIONS: Our findings emphasize the need for caution in interpreting TDP-43 overexpression data and stress the importance of controlling for exon skipping when generating models of TDP-43 proteinopathy.