Fig 2: TDP-43^ΔRRM1 impairs splicing repression by forming heterodimers with endogenous TDP-43.a Western blots after DSG crosslinking showing TDP-43^FL forms dimers, while N6A and 90–414 mutants reduce dimerization; dimer/monomer ratio quantified (n = 3, two-sided t-test). b Co-expression of TDP-43 mutants with PKmini showing TDP-43^F147/149 L and ΔRRM1 induce 5a1 insertion; PSI quantified (n = 3, one-way ANOVA). c Under shTDP-43, TDP-43^FL, F227/229 L and ΔRRM2 restore 5a1 suppression, while F147/149 L, ΔRRM1, N6A and 90–414 fail; PSI shown (n = 3, one-way ANOVA). d Endogenous PKN1-5a1 splicing is activated by ΔRRM1, but not FL or ΔRRM2; 5a1/ACTB ratio quantified (n = 3, one-way ANOVA). e ΔRRM1 induces 5a1 insertion in PKmini, but dimerization-deficient ΔRRM1^N6A does not (n = 3, one-way ANOVA). f BiFC imaging shows that prominent nuclear puncta are formed when TDP-43-VN173 and ΔRRM1-VC155 are co-expressed together with ΔRRM1, whereas no such puncta are observed when TDP-43-VN173 and TDP-43-VC155 are co-expressed with ΔRRM1. Scale bar, 10 µm. g Quantification of BiFC puncta per nucleus (n = 20 cells/group; two-sided Wilcoxon rank-sum test). h Working schematic: ΔRRM1 preferentially recruits TDP-43/ΔRRM1 heterodimers rather than TDP-43/TDP-43 homodimers. Source data are provided as a Source Data file.

Fig 3: TDP-43 binds UG-rich motifs near PKN1 exon 5a1 to suppress its inclusion.a Schematic of six candidate TDP-43 binding sites (site1–site6) between exons 4 and 5 of PKN1, ranked by CLIP-seq CITS scores. b RIP-qPCR (n = 3) and Western blots showing TDP-43 binding to the 5a1-containing RNA region. HC: heavy chain of IgG. c Minigene PKmini recapitulates TDP-43-dependent suppression of 5a1 inclusion; shTDP-43 induces 5a1 insertion (n = 3). d, e Deletion of site1–5 (PKmini^Δ1–5, d) increases 5a1 inclusion (e) (n = 3). f,g Site6 partially mediates residual TDP-43 repression upon site1–5 deletion. In the PKmini^∆1–5 reporter, shTDP-43 (f) enhances, overexpression (g) suppresses, 5a1 cryptic exon inclusion (n = 3). h,i Site6 is essential to prevent aberrant A3SS usage, and PKmini^Δ6 (h) shows exclusive selection of the A3SS (i) (n = 3). j,k TDP-43 knockdown (j) enhances A3SS usage in PKmini^Δ6, while TDP-43 overexpression (k) suppresses A3SS and restores constitutive splicing (n = 3). l Schematic of combinatorial site-deletion constructs. m A3SS PSI values across deletion backgrounds showing site3 and site5 cooperate with site6 to repress aberrant splicing (n = 3). n,o EMSA (o) showing direct TDP-43 binding to probes (n) covering site1–3, site5, and site6 (n = 3). p Working model: TDP-43 binding to multiple UG-rich elements suppresses 5a1 insertion and A3SS usage, loss of TDP-43 relieves this repression. Source data are provided as a Source Data file.

Fig 4: TDP-43 knockdown leads to production of a stable cryptic peptide, PKN207.a TDP-43 and UPF1 knockdown in HEK-293T cells; TDP-43 depletion upregulates PKN1-5a1, enhanced by combined UPF1 knockdown (n = 3, one-way ANOVA). b Western blots of stable shCTR and shTDP-43 cell lines showing reduced PKFL and a ~ 33 kDa band (red arrow) matching predicted PKN207 (n = 3). c Western blots shows PKN207 appears from 120 h post shTDP-43 transfection (n = 3, one-way ANOVA). d CHX chase assay showing PKN207 more stable than PKFL after tetracycline induction (n = 3; slope comparison by ANCOVA). e shPKN1 abolishes the ~33 kDa band in shTDP-43 cells, confirming its origin from PKN1, quantification shown (n = 3, one-way ANOVA). f Four PKN207 antibodies specifically recognize FLAG-PKN1^N207 without cross-reacting with PKN1^FL (n = 3). g All four antibodies detect endogenous ~33 kDa PKN207 in shTDP-43 cells; E29426 selected for subsequent use (n = 3). h Western blots showing PKN207 in the hippocampus of AD with pTDP-43 (AD1, AD5). i PKFL protein levels do not differ between Normal and AD groups (n = 5, two-sided t-test). j Paired-point plot showing elevated PKN207 in individuals with pTDP-43 pathology. k RT-PCR showing increased PKN1-5a1/PKN1 ratio in AD hippocampus (Normal n = 5, AD n = 6; two-sided Wilcoxon test). Source data are provided as a Source Data file.