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ScienceBiochemistryPeer-reviewed journal article

Short RNA chaperones promote aggregation-resistant TDP-43 conformers to mitigate neurodegeneration

Katie E. CopleyiD, Jocelyn C. MaunaiD, Helen L. DanielsoniD, Qizan CheniD, Busra OzguneyiD, Marilyn NgoiD, Longxin XieiD, Ashleigh SmirnoviD, Matt DavisiD, Leland C. MayneiD, Miriam LinsenmeieriD, Jack D. RubieniD, Cristian A BergmanniD, Bede PortziD, Bo Lim LeeiD, Hana M. OdehiD, Longsheng Lai, Yi‐Wei ChangiD, Martina HalleggeriD, Jernej UleiD, Piera PasinelliiD, Yan Poon, Jeetain MittaliD, Nicolas Lux FawziiD, Ben E. BlackiD, , James ShorteriD

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Abstract

Aberrant aggregation of the prion-like, RNA-binding protein TDP-43 underlies several debilitating neurodegenerative proteinopathies, including amyotrophic lateral sclerosis (ALS). Here, we define how short, specific RNAs antagonize TDP-43 aggregation. Short, specific RNAs engage and stabilize the TDP-43 RNA-recognition motifs, which allosterically destabilizes a conserved helical region in the prion-like domain, thereby promoting aggregation-resistant conformers. By mining sequence space, we uncover short RNAs with enhanced activity against TDP-43 and diverse disease-linked variants. The solubilizing activity of enhanced short RNA chaperones corrects aberrant TDP-43 phenotypes in optogenetic models and ALS patient-derived neurons. Remarkably, an enhanced short RNA chaperone mitigates TDP-43 proteinopathy and neurodegeneration in mice. Our studies reveal mechanisms of short RNA chaperones and pave the way for the development of short RNA therapeutics for fatal TDP-43 proteinopathies.

In-depth summary

Claude Opus 5.5 · xhigh effort · 5 Nov 2026 · from the full text of version 1 (15 Dec 2024) · CC BY-NC-ND 4.0

Short UG-rich RNAs bind and stabilize TDP-43 RNA-recognition motifs to allosterically destabilize its prion-like domain helical region, preventing aggregation and mitigating neurodegeneration in cells and mice.

Cytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 occur in most amyotrophic lateral sclerosis (ALS) and several other neurodegenerative proteinopathies, which currently lack effective therapeutics. While the 34-nucleotide RNA Clip34 was known to solubilize wild-type TDP-43 in vitro, its allosteric mechanism, activity against disease variants, and in vivo efficacy were unknown.

Key results

  1. ShownClip34 RNA binds cooperatively to the TDP-43 RNA-recognition motifs (RRMs) to allosterically inhibit prion-like domain (PrLD)-driven aggregation, while the PrLD conserved region (CR) negatively regulates RNA binding.

    Substoichiometric Clip34 (1:4 RNA:TDP-43) abolished wild-type TDP-43 aggregation (KD ~0.49 uM, Hill slope ~2.4) but failed to inhibit RNA-binding-deficient TDP-43^5FL or TDP-43 lacking both RRMs; deleting the PrLD or its alpha-helical CR increased Clip34 binding affinity by ~1.5-fold (KD ~0.32 uM) and ~2.1-fold, respectively. · Clip34 is an allosteric antagonist of TDP-43…; Revealing allosteric crosstalk between the…; Figure legends

  2. ShownHydrogen/deuterium-exchange mass spectrometry (HXMS) demonstrates that Clip34 binding stabilizes both TDP-43 RRMs while allosterically destabilizing the transient alpha-helix in the PrLD CR.

    Across 1 s to 14.5 h HXMS timepoints (>=87.9% sequence coverage), Clip34 decreased deuterium exchange throughout RRM1 and RRM2 (including Y155-D174, F149, F229, F231) while increasing early-timepoint (1-18 s) exchange across CR residues M323-L340 (especially Q331 and S332) and depopulating the slow-exchanging bimodal CR state. · Clip34 binding remodels TDP-43 by stabilizing…; Hydrogen/deuterium-exchange mass spectrometry…; Figure legends

  3. ShownEngineered Clip34_UG6 and natural MALAT1-derived Malat1_start short RNAs potently inhibit aggregation across diverse ALS/FTD-linked TDP-43 missense and post-translational modification mimetic variants.

    Clip34 inhibited wild-type and mutant TDP-43 (IC50 ~0.12-0.69 uM) but was less active against the lysine acetylation mimic TDP-43^(K145/192Q) (IC50 ~0.69 uM vs ~0.5 uM for WT); Clip34_UG6 (IC50 ~0.16-0.45 uM, ~0.35 uM for K145/192Q) and Malat1_start (IC50 ~0.36 uM for WT, ~0.17-0.44 uM across variants) overcame this deficit. · Enhancing Clip34 activity against diverse…; Defining additional potent short RNA chaperones…; Effective RNA chaperones can engage the TDP-43…; Figure legends

  4. ShownMalat1_start reduces cytoplasmic TDP-43 inclusions in optogenetic human cells without impairing splicing function and restores nuclear TDP-43 localization in C9orf72-ALS patient-derived neurons.

    In optoTDP-43 HEK293 cells, 500 nM Malat1_start and (UG)17 significantly reduced cytoplasmic puncta area per cell versus control RNA (n=3 biological replicates), but (UG)17 impaired TDP-43 splicing in the CUTS biosensor assay whereas Malat1_start and Clip34 did not and restored the nuclear/cytoplasmic TDP-43 ratio in C9-ALS iPSC-derived neurons. · Short RNAs reduce cytoplasmic TDP-43 aggregation…; Clip34 and Malat1_start RNAs do not cause TDP-43…; Short RNAs restore physiological TDP-43…; HEK293 cell oligonucleotide treatment

  5. ShownIntraspinal delivery of 2'OMe-modified Malat1_start reverses cytoplasmic TDP-43 aggregation and prevents motor neuron loss in an acute AAV9-TDP-43^DeltaNLS mouse model.

    Administering 100 ug Malat1_start at day 7 post-AAV9 injection maintained ventral horn ChAT+ motor neurons at 73.9% (day 10), 72.2% (day 12), and 58.1% (day 14) of sham controls versus 40.1%, 31.0%, and 28.7% in saline controls, while reducing average TDP-43 puncta size at days 10 and 12. · Malat1_start mitigates neurodegeneration and…; Spinal delivery of sRNA; Figure legends

Methods, data and reproducibility

Purified wild-type, domain-deletion, missense, and PTM-mimetic TDP-43-MBP-His constructs to assay TEV-protease-induced aggregation turbidity, electrophoretic mobility shifts, fluorophore-quencher stem-loop unfolding, HXMS, and solution NMR with short RNAs. Evaluated lead 2'OMe-modified RNAs in optoTDP-43 and CUTS HEK293 cells, C9-ALS iPSC-derived neurons, and intraspinal AAV9-TDP-43^DeltaNLS mice.

  • Recombinant human TDP-43 domain deletions, ALS/FTD missense mutants, and PTM mimetics expressed in E. coli for turbidity, EMSA, HXMS, and HSQC NMR · human · n = 3-8 replicates per assay; >=87.9% HXMS coverage · BMRB ID 27613
  • OptoTDP-43 and CUTS biosensor stable HEK293 cell lines · human · n = n=3 biological replicates (20 fields of view per well)
  • Healthy control (CS15iCTR-5) and C9orf72-ALS patient-derived iPSC motor neurons (CS29iALS-n1, CS52iALS-n6A, JH034) · human · n = 4 iPSC lines (n=3 biological replicates, 20 images/group)
  • Female C57BL/6J mice (p180) injected intraspinally with AAV9-TDP-43^DeltaNLS and treated with saline or Malat1_start RNA · mouse · n = 26 mice total (20 in main cohort: n=2 sham, n=9 saline, n=9 Malat1_start)
Methods & tools
SEQUEST Proteome Discoverer, HDExaminer, ExMS2, MATLAB, Bruker TopSpin, NMRPipe, CCPNMR, RNAstructure, NIS-Elements, Imaris, GraphPad Prism, Image Studio Lite
Reagents & environment
NIS-Elements AR1 Analysis 4.51; Imaris Imaging Software (Version 10.1.0) (S39, S47).
Reproducibility gaps
All data are available only upon request from the lead contact, and no original code is reported (S17).
Limitations (2)
  • The synthetic repetitive RNA (UG)17, despite potent anti-aggregation activity, interfered with endogenous TDP-43 splicing function in the CUTS biosensor assay and was unsuitable for further development (S11, S14). · stated by the authors
  • In the mouse AAV9-TDP-43^DeltaNLS model, only female mice were tested to control starting weight variance, and by day 14 after a single Malat1_start dose, TDP-43 puncta numbers per neuron began to increase again (S13, S18). · stated by the authors

Read this if you study RNA-binding protein phase separation, TDP-43 structural dynamics, or oligonucleotide therapeutics for amyotrophic lateral sclerosis and related proteinopathies.

Citations
20
FWCI
61.61
Field percentile
top 1%

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