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

Abstract

The loss of nuclear TDP-43 localization and its accumulation in cytoplasmic aggregates are hallmarks of neurodegeneration and major therapeutic targets. We recently demonstrated that TDP-43 binding to endogenous nuclear GU-rich RNAs sequesters TDP-43 in the nucleus and restricts 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 and identified (GU)16 as a strong multivalent binder. When transfected into cells, unlike monovalent oligonucleotides that displaced TDP-43 from the nucleus, (GU)16 preserved steady-state TDP-43 nuclear localization and prevented transcriptional blockade-induced TDP-43 mislocalization. RNA pulldowns from (GU)16-transfected cells confirmed that (GU)16 induced high molecular weight RNP complexes, incorporating TDP-43 and possibly other GU-binding proteins. Transfected (GU)16 caused partial failure of TDP-43 cryptic exon repression, likely because the high-affinity oligonucleotides diverted TDP-43 from endogenous RNAs. Thus, while GU-rich oligonucleotides can attenuate TDP-43 mislocalization, optimization is needed to avoid TDP-43 loss of function.

CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

“…This is supported by the comparison of CUTS with the CFTR-TS or UNC13A-TS cassette (Figure 1), underscoring the potential of CUTS to outperform single minigene-based approaches in TDP-43 LOF detection. While recent work in two recent preprints suggests other CE biosensors are in development, CUTS appears to exhibit enhanced sensitivity (Wilkins et al, 2023;Zhang et al, 2023). The expression of the GFP reporter in CUTS achieved up to 118,224-fold increase upon TDP-43 knockdown, compared to ADNP2 (< 5-fold) (Zhang et al, 2023); TDP-REGv1 (<20-fold); and TDP-REGv2 (<300-fold) (Wilkins et al, 2023).…”

“…While recent work in two recent preprints suggests other CE biosensors are in development, CUTS appears to exhibit enhanced sensitivity (Wilkins et al, 2023;Zhang et al, 2023). The expression of the GFP reporter in CUTS achieved up to 118,224-fold increase upon TDP-43 knockdown, compared to ADNP2 (< 5-fold) (Zhang et al, 2023); TDP-REGv1 (<20-fold); and TDP-REGv2 (<300-fold) (Wilkins et al, 2023). We also show that CUTS can detect ultra-low levels of TDP-43 knockdown (increasing > 7-fold), below the WB or RT-qPCR detection limit.…”