TDP-43 directly inhibits RNA accumulation in neurites through modulation of RNA stability

Charlie Moffatt, Ankita Arora, Katherine F. Vaeth, Bryan B. Guzman, Gurprit Bhardwaj, Audrey Hoelscher, Levi B. Gifford, Holger A. Russ, Daniel Dominguez, J. Matthew Taliaferro

Posted on: 10 April 2025 , updated on: 11 April 2025

Preprint posted on 1 March 2025

An intriguing twist! TDP-43 (linked to ALS) keeps RNAs out of neuronal projections by making them unstable preventing RNA buildup in neurites

Selected by Felipe Del Valle Batalla

Categories: cell biology, neuroscience

Background

Neurons, with their elongated shape and long axonal distances, heavily rely on RNA localization to establish their subcellular proteomes. Disruptions in RNA localization, often due to defect RNA-binding proteins (RBPs), have been implicated in various neurological diseases [1]. Of these RBPs, TAR DNA binding protein 43 (TDP-43) has been extensively studied due to its strong association with amyotrophic lateral sclerosis (ALS) [2]. What is known already is that TDP-43 promotes RNA transport to neuronal projections. However, the specific RNA targets and the underlying mechanisms remained largely unknown, prompting the authors of this preprint to investigate this.

Main findings

TDP-43 loss leads to increased RNA accumulation in neurites

Moffatt and colleagues began by investigating the role of TDP-43 in RNA localization to neuronal projections. To this end, they used microporous transwell membranes to separate neuronal cell bodies from neurites (Fig.1A) and quantified RNA abundance in each compartment using high-throughput sequencing (Fig.1C). They generated TDP-43 knockout cell lines using a doxycycline-off system and upon TDP-43 depletion, they observed that hundreds of RNAs showed increased accumulation in neurites compared to control cells. This suggested that TDP-43 acts to keep these RNAs out of neuronal projections.

Fig.1 in the preprint. (A) Schematic of subcellular fractionation and RNA isolation setup. (C) Changes in RNA localization to neurites in CAD cells upon loss of TDP-43. Figures made available under a CC-BY-ND 4.0 International license.

The authors then selected and validated candidate RNAs by single molecule RNA FISH and confirmed that some RNAs became significantly more enriched in neurites upon TDP-43 loss, supporting the findings from the subcellular fractionation and RNA-seq experiments. (Fig.2 A)

TDP-43 localization targets are enriched in TDP-43 binding motifs

Next, the authors analyzed the 3′ UTR sequences of the RNAs that showed increased neurite localization in TDP-43 KO cells and found that they were highly enriched in known TDP-43 binding motifs. To test if the 3′ UTRs were responsible for the TDP-43-dependent localization changes, 3′ UTRs from three TDP-43-sensitive RNAs (Diras1, Ksr2, Wasf3) were fused to a reporter transcript (Fig.3D). The 3′ UTRs were sufficient to drive TDP-43-dependent localization of the reporter, causing it to become more neurite-enriched upon TDP-43 loss. This indicated that these 3′ UTRs contain elements that mediate RNA localization regulated by TDP-43.

To detect the specific sequences within these 3′ UTRs responsible for TDP-43 regulation, a massively parallel reporter assay (MPRA) was designed (Fig.4A). This assay simultaneously tested the ability of thousands of 260nt RNA sequences distributed across different 3′ UTRs to mediate TDP-43-dependent RNA localization. The MPRA identified unique 260nt RNA sequences that were sufficient to make the localization of a reporter RNA TDP-43-sensitive.

Fig. 3D and Fig.4A in the preprint. (D) Differences in neurite localization for a reporter transcript containing the indicated 3′ UTRs in wildtype and TDP-43 knockout cells. (A) Design of MPRA for the interrogation of TDP-43-mediated RNA localization. Figures made available under a CC-BY-ND 4.0 International license.

RNA secondary structure plays a role in TDP-43 occupancy and function

To directly assess TDP-43 binding to the identified RNA sequences, RNA Bind-n-seq (RBNS) was performed using purified recombinant TDP-43 (Fig.5A). Consistent with the MPRA results, the RBNS data showed that oligos containing TDP-43 motifs located within CLIP-seq peaks exhibited a much higher affinity for TDP-43 (Fig.5C).

Fig.5 in the preprint. (A) Schematic of RNA-Bind-n-seq assay used to determine relative affinities for TDP-43. (C) Differences in TDP-43 affinity for RNA sequences that contain TDP-43 motifs and their companion sequences in which those motifs were mutated. Figures made available under a CC-BY-ND 4.0 International license.

TDP-43 regulates RNA localization by modulating RNA stability and inhibits RNA localization to neurites in human motor neurons.

To understand the mechanism by which TDP-43 prevents neurite accumulation of its target RNAs, the stability of the reporter RNAs used in the MPRA was measured using SLAM-seq. The results show that reporter oligos containing TDP-43 motifs within CLIP-seq peaks displayed significantly increased stability upon TDP-43 loss, indicating that TDP-43 normally promotes the turnover of these RNAs (Fig.6C). This suggests that TDP-43 binds to unstructured motifs and destabilizes target RNAs, preventing its accumulation in neurites.

To further validate the findings in a more physiologically relevant system for humans, RNA localization patterns were examined in human iPSC-derived motor neurons following TDP-43 KO. Consistent with the previous findings, RNAs that became more neurite-enriched after TDP-43 loss in human motor neurons had significantly more TDP-43 motifs in their 3′ UTRs (Fig.7A), confirming the role of TDP-43 as a negative regulator of RNA abundance in neurites. Finally, CAD cell lines expressing either wildtype TDP-43 or the ALS-associated Q331K mutant TDP-43 were created. What was observed next is that RNAs that became more neurite-enriched in cells expressing the Q331K mutant were the same RNAs that were more neurite-enriched in TDP-43 knockout cells (Fig.7D). This suggests that the Q331K mutation leads to a loss of TDP-43’s ability to regulate RNA localization, potentially contributing to RNA mislocalization in ALS patients.

Why I think this work is relevant

I highlighted this preprint because it reveals that the ALS-associated RNA binding protein TDP-43 primarily functions to prevent RNAs from entering neuronal projections by promoting their instability, a finding that diverges from some previous studies. A notable strength of this work lies in its integrated approach, which integrates subcellular fractionation, single-molecule imaging, and quantitative high-throughput assays (MPRA, RBNS, and SLAM-seq) on the same set of RNA sequences. This comprehensive approach enabled the researchers to establish a direct link between TDP-43 binding, RNA stability, and RNA localization.

Question for the authors

While you focus on the general trend of TDP-43 acting to keep RNAs out of neuronal projections, are there specific functional categories or pathways that are particularly enriched among the mislocalized RNAs that could provide insights into the specific cellular processes disrupted in neuronal projections due to TDP-43 dysfunction?

References

[1] Farris S, Hacisuleyman E, Donlin-Asp P and Cioni J-M (2022) Editorial: RNA Localization and Localized Translation in Neurons. Front. Integr. Neurosci. 15:831038. doi: 10.3389/fnint.2021.831038

[2] Jo, M., Lee, S., Jeon, YM. et al. The role of TDP-43 propagation in neurodegenerative diseases: integrating insights from clinical and experimental studies. Exp Mol Med 52, 1652–1662 (2020). https://doi.org/10.1038/s12276-020-00513-7

Tags: rna, tdp-43

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Author's response

Matthew Taliaferro, PhD shared

The three genes we found to show the strongest increases in neurite-enrichment (Diras1, Ksr2, and Wasf3), are all related to Ras signaling. There is some literature connecting dysregulated Ras signaling to neurodegeneration. So there may be a link there between these findings and ALS phenotypes, but this is very speculative.

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