TDP-43 nuclear loss in FTD/ALS causes widespread alternative polyadenylation changes

In frontotemporal dementia and amyotrophic lateral sclerosis, the RNA-binding protein TDP-43 is depleted from the nucleus. TDP-43 loss leads to cryptic exon inclusion but a role in other RNA processing events remains unresolved. Here, we show that loss of TDP-43 causes widespread changes in alternative polyadenylation, impacting expression of disease-relevant genes (e.g., ELP1, NEFL, and TMEM106B) and providing evidence that alternative polyadenylation is a new facet of TDP-43 pathology.


Main
TDP-43 binds to uridine/guanine (UG)-rich motifs in RNA transcripts 1,2 and plays a critical role in various aspects of RNA metabolism [3][4][5] .Defects in RNA metabolism are considered central to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) pathogenesis 3 .However, it is not yet fully understood what genes TDP-43 regulates, what aspect of their RNA metabolism TDP-43 regulates, and how their dysregulation promotes neurodegeneration.A major role of TDP-43 has emerged as a repressor of so-called cryptic exons during splicing 6 .Cryptic exons reside in introns of genes and are normally excluded from mature mRNAs.
Besides its well-established role in splicing, TDP-43 also plays a critical role in other aspects of RNA processing.Do some of these additional TDP-43-dependent RNA processing pathways also contribute to disease?One potential disease-relevant RNA processing pathway is alternative polyadenylation (APA) -a major layer of gene regulation that occurs in >60% of human genes 17 .When a gene is transcribed into mRNA, it is cleaved and polyadenylated, which functions to stabilize the mRNA, facilitate its nuclear export, and regulate its translation 18 .If alternative polyadenylation (polyA) sites are utilized, it could produce mRNA isoforms that have different 3' untranslated region (UTR) lengths, impacting RNA/protein levels, subcellular localization, or even protein functions 17,19 .If APA occurs prematurely, it can truncate mRNAs, reducing full-length protein levels 17,19 .Previous genome-wide TDP-43 binding studies revealed that TDP-43 binding is enriched not only in introns of genes but also in 3' UTRs 1,2 , suggesting a role in regulating polyadenylation.Indeed, TDP-43 knockdown or disease-associated TDP-43 mutations affect polyadenylation [20][21][22] , including its own polyadenylation 2,23 and premature polyadenylation in STMN2 7,8 .Notably, widespread APA changes have been observed in ALS patient samples using bulk RNA-sequencing (RNA-seq) 24 and single nucleus RNA-seq 25 , although it is unclear whether these changes are directly owing to TDP-43 dysfunction.
To test the hypothesis that APA caused by TDP-43 dysfunction contributes to FTD/ALS, we first performed APA analysis in an RNA-seq dataset 26 , in which neuronal nuclei with and without TDP-43 were sorted from FTD/ALS postmortem brain samples for RNA-seq (Fig. 1a, top panel).We recently re-analyzed this dataset to discover TDP-43-dependent cryptic splicing events 11 .Here, we 're-re-analyzed' it to search for APA events (Fig. 1a, bottom panel).We used two different APA analysis programs and identified 41 APA changes using APAlyzer 27 and 62 APA changes using QAPA 28 (Fig. 1b, S1a; Table S1; adjusted p value < 0.1).
Only two genes with significant APA changes were common between both programs: LRFN1 and MARK3 (see also Fig. S2k and Arnold et al. 29 ), likely because of different polyA databases used by each program and the low resolution of RNA-seq for APA analysis.We thus considered APA changes identified by either program, which revealed APA events in genes critical for neuronal function, such as LRFN1 and SYN2.Some of these APA events are associated with gene expression changes (Fig. S1b), likely impacting their corresponding protein levels (see below).
To comprehensively map TDP-43-dependent APA events and define how TDP-43 dysfunction contributes to neurodegeneration via APA, we sought a more sensitive assay than RNA-seq for APA analysis, because RNA-seq cannot map novel polyadenylation events, identify actual polyA sites, or capture 'internal' premature polyadenylation events (i.e., ones that truncate the RNA/protein).We used a specialized transcriptomic method, 3' end-seq, to map polyA sites with single-nucleotide resolution in iNeurons with or without TDP-43 knockdown (Fig. 2a, 1d).We obtained a total of 117,823 putative polyA sites with a known upstream polyA signal, of which 46,760 sites are novel (Fig. S2a), when compared to a compendium of polyA site annotations 31 .Consistent with previous findings 2,20 , the majority of polyA sites we identified are in 3' UTRs (Fig. S2b).3' end-seq confirmed that loss of TDP-43 lengthened the 3' UTR of LRFN1 (Fig. 2b, top panel), which we also observed in our analysis of FTD/ALS postmortem brain samples (Fig. 2b, top panel; Fig. 1b).
Notably, 3' end-seq also captured premature polyadenylation in STMN2, which was previously identified but not apparent in standard RNA-seq data (Fig. 2b, bottom panel), demonstrating the power of 3' end-seq for studying premature polyadenylation, the most detrimental form of APA since it truncates the RNA/protein.
In total, TDP-43 knockdown altered the usage of 8,169 polyA sites (|∆PUI| > 0.1 and adjusted p value < 0.05 from LAPA, a 3' end-seq data focused program) and caused APA changes in 2,220 genes (Fig. 2c; Table S3).By cross-referencing APA sites with a curated genome-wide TDP-43 binding site dataset 32 , we found that ~70% of genes with APA events have at least one TDP-43 binding site, consistent with a direct role of TDP-43 in regulating APA of these genes.Like recent findings in ALS samples 24 and FTD samples (Fig. 1b, S1a) as well as our RNA-seq data from iNeurons (Fig. 1e), the majority of APA changes (1,881) detected by 3'end-seq lengthened RNA transcripts; 340 APA events were associated with at least a 1.5-fold change in RNA level (Fig. 2d), suggesting that APA changes could alter gene expression.Notably, several of these APA changes were also observed in FTD/ALS postmortem brain samples and were in genes connected to ALS or FTD (Table S4; see below).
To define whether polyA site strength influences TDP-43 regulated APA, we estimated the probability of cleavage and polyadenylation for each identified polyA site using a deep residual neural network-based model, Aparent2 33 , that accurately predicts polyA site usage.Interestingly, we found that polyA sites with reduced usage upon TDP-43 knockdown are weaker than the ones with increased usage upon TDP-43 knockdown (Fig. 2e), indicating that TDP-43 either promotes the usage of weak polyA sites or suppresses the usage of strong ones.Whereas genes with significant 3' UTR shortening show no difference in polyA strength between proximal and distal polyA sites, genes with significant 3' UTR lengthening have stronger distal sites compared to proximal ones (Fig. 2f).Given the co-transcriptional nature of cleavage and polyadenylation, our findings suggest that in the case of 3' UTR lengthening upon TDP-43 knockdown, a stronger distal polyA site might compensate for its positional disadvantage over the proximal polyA site during transcription and therefore get activated upon TDP-43 knockdown.The impact of polyA site strength on shaping TDP-43-regulated APA provides a toehold to prioritize antisense oligonucleotide-based therapeutic strategies to target specific APA events akin to current approaches underway to target cryptic splicing events 15 .
3' end-seq also empowered the discovery of "cryptic" polyA sites (ones not used under normal conditions but revealed upon TDP-43 knockdown).TDP-43 knockdown activated 457 cryptic polyA sites in 424 genes (Table S5); 163 cryptic polyA sites occurred downstream of a gene's annotated 3' end, such as the FTD risk factor RFNG 34 (Fig. S2c), SIX3, TLX1, and ELK1 (Fig. S2d-f, and see also Bryce-smith et al. 35 ) and 153 cryptic events induced premature polyadenylation in genes such as TNIP1, EGFR, SLC24A3, and GSTO2 (Fig. S2g-j).Like coordinated activation of splicing and polyadenylation in intron 1 of STMN2, we observed coordinated activation in PIGL, HNRNPA1, and ARHGAP32 (Fig. 2g), the latter of which is observed by Fratta and colleagues 35 .Cryptic splicing of ARHGAP32 was recently reported 12 , and we also confirmed this in RNAseq data from FTD/ALS postmortem brain samples (Fig. 2g).These results suggest a potential coupling between cryptic splicing and the activation of cryptic polyadenylation sites.TDP-43 depletion increased the usage of two distal polyA sites in ELP1 that lengthened its 3' UTR (Fig. 3a, left panel; Fig. S3a) and such lengthening is present across different datasets (Fig. S3b; see also Arnold et al. 29 ).ELP1, also called IKBKAP, encodes a subunit of the elongator complex, which has been functionally and genetically linked to ALS [36][37][38] .In addition to ELP1, we observed APA changes in two other subunits of the elongator complex (ELP3 and ELP6) upon TDP-43 knockdown (Fig. S3c, S3d).These APA changes are associated with protein levels; TDP-43 knockdown led to increased protein levels of ELP1 and ELP3 (Fig. 3a,   S3c).Together with a recent finding of reduced aminoacylation of tRNA Phe in FTD/ALS 39 , our observations suggest that altered tRNA metabolism might be an important mechanism contributing to FTD/ALS.Previous studies found that TDP-43 directly binds to the 3' UTR of NEFL to stabilize its RNA levels 40 .
NEFL encodes neurofilament light chain (NF-L), which has emerged as a sensitive prognostic biomarker for diverse neurodegenerative diseases 41 , including FTD/ALS 42 .We observed three major polyA sites in NEFL, with the most distal one being the most frequently used (Fig. 3b, left panel).TDP-43 knockdown not only reduced NEFL RNA levels (Fig. S3e), consistent with previous findings 43 , but also further shifted the apparent polyA site usage from proximal to distal (Fig. 3b, left and mid panels; Fig. S3f) and reduced NF-L protein levels (Fig. 3b, right panel).These observations suggest that together with NEFL 3' UTR-targeting microRNAs 44 , loss of TDP-43 induced APA changes might further reduce NF-L levels.A direct role of TDP-43 in regulating NF-L levels adds complexity to the use of this biomarker in TDP-43 proteinopathies.
TDP-43 knockdown also shifted polyA site usage from proximal to distal in another FTD/ALS-linked gene, SFPQ (Fig. 3c; Fig. S3g).SFPQ encodes a ubiquitously expressed RNA-binding protein that plays key roles in RNA metabolism 45,46 .SFPQ encodes two protein isoforms; the shorter isoform uses a stop codon downstream of the proximal polyA site (Fig. 3c) and lacks a nuclear localization signal 45 .Intriguingly, depletion of SFPQ from the nucleus has been observed in sporadic ALS spinal cord 47 , and its interaction with FUS, another FTD/ALS risk gene, is impaired in neuronal nuclei in FTLD-TDP 48,49 .Thus, loss of TDP-43 promotes the usage of SFPQ's distal polyA site (Fig. 3c), which might upregulate the shorter NLS-lacking SFPQ isoform and contribute to its nuclear depletion in disease.
The sensitivity of 3' end-seq further revealed that TDP-43 knockdown lengthened TMEM106B's 3' UTR (Fig. 3d), which we also confirmed by qRT-PCR (Fig. S3h).TMEM106B is a top genetic risk factor that emerged in a genome-wide association study for FTLD-TDP 50 .C-terminal fragments of TMEM106B were recently found to form amyloid fibrils in brains of older individuals and patients with neurodegenerative disorders [51][52][53][54] .By targeted analysis of RNA-seq read coverage across the TMEM106B 3' UTR, we confirmed that loss of TDP-43 is associated with a longer TMEM106B 3' UTR in FTD/ALS postmortem brain samples (Fig. 3e).We also analyzed the TMEM106B 3'UTR in a series of 83 frontal cortex brain samples from the Mayo Clinic Brain Bank using qRT-PCR and found a significant increase in longer TMEM106B 3' UTRs in the frontal cortices of patients with FTLD-TDP compared with healthy controls (Fig. 3f), indicating that the increased 3' UTR length of TMEM106B might be functionally relevant in FTD/ALS.Because TMEM106B protein levels are altered in disease 50,55 , we next asked whether TDP-43 knockdown affected TMEM106B protein levels.Using a condition that has been shown previously to preserve TMEM106B dimers on SDS-PAGE 55 (see also Fig. S3i), we found that TDP-43 knockdown did not affect levels of TMEM106B monomers (~42 KDa) but reduced dimer levels (Fig. 3g).A recent proteomics study also detected decreased TMEM106B levels caused by TDP-43 knockdown 12 .TDP-43 knockdown only modestly reduced TMEM106B RNA levels (Fig. S3j), suggesting that the longer 3' UTR might affect TMEM106B protein levels via translation.To test this hypothesis, we cloned short and long versions of the TMEM106B 3' UTR into a dual-luciferase reporter.We mutated the proximal polyA site in the long 3' UTR-containing reporter to prevent its usage and confirmed the production of the intended long 3' UTR (Fig. S3k).The long 3' UTRcontaining reporter had significantly lower luciferase activity and higher RNA levels than the short 3' UTRcontaining reporter (Fig. 3h), providing evidence that the loss of TDP-43-induced longer TMEM106B 3' UTR reduces translation efficiency.But how could this reduce the formation of TMEM106B dimers?TMEM106B APA might impact dimer levels by influencing the subcellular localization of TMEM106B mRNA and its ability as a scaffold for protein-protein interactions, as has been shown for other APA events 17 .Recent studies discovered an Alu element insertion in the TMEM106B 3' UTR, in perfect linkage with the top FTLD-TDP risk allele at this locus [56][57][58] .Given the proximity of these variants to TMEM106B's distal polyA site, future work will explore if and how these disease-associated genetic variants impact APA.
In the present study, we applied 3' end-seq to comprehensively map neuronal polyadenylation on a transcriptomic scale with base-pair resolution and discovered that TDP-43 dysfunction causes widespread APA changes, some of which are in genes critical for neuronal function and in disease-associated genes.These changes are highlighted by coupled cryptic splicing and premature polyadenylation in ARHGAP32 (Fig. 2g), changes observed in multiple subunits of the elongator complex (Fig. 3a, S3c, S3d), and 3' UTR lengthening in NEFL, SFPQ, and TMEM106B (Fig. 3b-d).This study is accompanied by two independent manuscripts also presenting widespread APA changes associated with TDP-43 dysfunction in FTD/ALS (Fratta and colleagues 35 ; La Spada and colleagues 29 ).Despite using different approaches, each of the three studies observed common APA changes (Fig. 2g, S2d-f, S2k-m), underscoring the importance of APA changes during neurodegeneration.
Our application of 3' end-seq complements and extends RNA-seq based APA analysis because it enables de novo identification of polyadenylation events with base-pair resolution, revealing complex and highly sensitive APA changes (e.g., NEFL, ELP3, and TMEM106B).Together, the findings of these three studies provide evidence that TDP-43 pathology contributes to disease pathogenesis through not only cryptic splicing but now also changes in alternative polyadenylation.level changes on the y-axis, illustrating that APA changes could alter RNA levels.Genes with significant APA changes are plotted; genes with significant RNA level changes are labeled in red when favoring distal polyA sites or blue when favoring proximal polyA sites.e, Cumulative plots show that for TDP-43 regulated APA sites, distal polyA sites are stronger than proximal polyA sites.f, Cumulative plots show that for genes with significant 3' UTR lengthening upon TDP-43 KD, their distal polyA sites are stronger than their proximal ones.PolyA site scores (in panels e and f) were calculated using Aparent2, expressed as log odds ratio, and reflect polyA site strengths.g, TDP-43 KD activated cryptic splicing and premature polyadenylation in intron 12 of ARHGAP32.The "polyA DB" track marks annotated polyA sites 59 and the "TDP-43 sites" track marks the observed TDP-43 binding 32 .Gene structure is shown on the bottom.a, TDP-43 knockdown (KD) lengthened the 3' UTR of ELP1 (left panel) and increased ELP1 protein levels (middle and right panels).b, TDP-43 KD down-regulates the usage of proximal polyA site usage of NEFL, leading to increased usage of the most distal polyA site (left two panels) and reduced protein levels (right two panels).c, TDP-43 KD shifted the polyA site usage from proximal to distal in SFPQ.The gene structure shows that the top three RNA isoforms produce a long protein isoform, and the bottom RNA isoform produces a short protein isoform without NLS.d, TDP-43 KD lengthened the 3' UTR of TMEM106B.e, Targeted APA analysis of TMEM106B using RNA-seq from FTD/ALS brain samples.f, Level of the long TMEM106B 3' UTR is significantly increased in the frontal cortices of FTLD-TDP patients compared to healthy controls.P-values were calculated by two-tailed Mann-Whitney test.g, WB shows reduced TMEM106B dimer levels after 12-day TDP-43 KD. h, Presence of the long TMEM106B 3' UTR reduces translation efficiency.Bar plots show that longer TMEM106B 3' UTR led to lower luciferase activity (top panel) and higher RNA levels (bottom panel).No insert, a dual-luciferase reporter without TMEM106B 3' UTRs; short, a dual-luciferase reporter with the short TMEM106B 3' UTR; long, a dual-luciferase reporter with the long TMEM106B  a, Volcano plot shows that QAPA, another popular APA analysis program, also uncovered that loss of TDP-43 is associated with APA changes.The log2(fold change) represents the difference of distal polyA site usage between TDP-43 negative nuclei and TDP-43 positive nuclei.Adjusted p-values were calculated by DEXSeq.b, Volcano plot shows that genes have both APA changes and RNA level changes.Only genes with significant APA changes, detected by APAlyzer or QAPA, are plotted with RNA level change on the x-axis and the corresponding adjusted p value on the y-axis.Differential RNA expression was analyzed using DESeq2.c-e, Volcano plots show that APAlyzer-identified APA changes upon TPD-43 knockdown (KD) in iMNs (c), in iNeurons carrying K263E mutation in TDP-43 (d), and in iMNs carrying M337V mutation in TDP-43 (e).Details, as in Fig. 1e.

Immunoblotting of TMEM106B
After 12 days of TDP-43 knockdown, cells were lysed and normalized as described above.To detect the impact of TDP-43 knockdown on the dimer level of TMEM106B, normalized cell lysates were mixed with 2x laemmli buffer (Bio-Rad) containing 5% 2-mercaptoethanol (Sigma-Aldrich) and loaded directly onto 4-20% Tris-Glycine mini gels (Thermo Fisher XP04205BOX) for gel electrophoresis on ice.To detect the temperature sensitivity of TMEM106B dimer, cell lysates were incubated at 4˚C, 37˚C, 70˚C, and 85˚C for 10 min and then loaded onto a 4-20% Tris-Glycine mini gel for gel electrophoresis on ice.After electrophoresis, samples were transferred onto 0.45-μm PVDF membranes (Bio-Rad 162-0115) at 250 mA for 2 h using the wet transfer method (Bio-Rad Mini Trans-Blot Electrophoretic Cell 170-3930).Membranes were blocked in 5% non-fat dry milk in TBST for 1 h and then incubated overnight at 4˚C in blocking buffer containing the antibody against TMEM106B (1:500, Cell Signaling Technology 93334).The secondary antibody, imaging, and quantitation are the same as described above.

Total RNA extraction from iNeurons
Total RNA was extracted using Trizol according to the manufacturer's instructions.Total RNA was then treated with Turbo DNase (Thermo Fisher AM2238) and cleaned by Zymo's clean and concentration columns.The quality of RNA was examined by on a High Sensitivity RNA ScreenTape (Agilent, Tapestation).

qRT-PCR from iNeurons
Total RNA (500 ng) was reverse transcribed to cDNA using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, RR047A).qPCR was carried out using PowerUp™ SYBR™ Green Master Mix kit and detected by QuantStudio3 system (Thermo Fisher).Primers are listed in Table S6.

Total RNA sequencing
Total RNAs from scramble shRNA or TDP-43 shRNA treated cells were used to construct RNA-sequencing libraries using the SMARTer Stranded Total RNA-Seq Kit v2 -Pico Input Mammalian kit (Takara 634411), according to the manufacturer's instructions.The resulting libraries were quantitated, pooled, and sequenced on a Nextseq 500 machine using the 150-cycle high output kit in a 75bp paired-end mode (Illumina).

Gene expression analysis
Adaptors in FASTQ files were trimmed using fastp.The adapter trimmed FASTQ files were used for differential gene expression analysis using Salmon and DESeq2.

Splicing analysis
The adapter-trimmed FASTQ files were mapped to human genome (hg38) following ENCODE recommended settings using STAR.The unique-mapping, properly paired reads were then used for splicing analysis using leafcutter.Cryptic splicing events were called by leafcutter.

APA analysis using RNA-seq data
Both in-house RNA-seq and publicly available datasets were used for the analysis.The publicly available datasets were downloaded from GEO or SRA (GSE126542, GSE121569, GSE147544, GSE196144, and ERP126666).The adapter trimmed FASTQ files were mapped to human genome (hg38) following ENCODE recommended settings using STAR.The unique-mapping, properly paired reads were then used for APA detection by either APAlyzer or QAPA and then differential analysis by DEXseq.APAlyzer uses polyA DB3 (https://exon.apps.wistar.org/PolyA_DB/)and QAPA uses PolyASite 2.0 (https://polyasite.unibas.ch/)as polyA site reference for detecting APA events.

3'end-seq
To comprehensively map polyadenylation and quantify alternative polyadenylation, three control samples and three TDP-43 knockdown samples were used for 3' end-seq.For each sample, 500 ng of total RNA was used to construct the 3' end-seq library using Quantseq REV kit from Lexogen, according to the manufacturer's instructions.The resulting 3' end-seq libraries were quantified by Qubit, checked for library sizes on a D1000 high sensitivity chip (Agilent Technologies, Tapestation), pooled, and sequenced with on a Nextseq 500 or Nextseq 2000 machine using the 150-cycle high output kit in a 75bp paired-end mode (Illumina).

APA analysis using 3' end-seq data
The sequenced 3' end-seq libraries were adapter-trimmed, and quality filtered using bbduk.The filtered reads were mapped to human genome (hg38) using STAR and then extracted for unique-mapping, properly paired reads using Samtools.If a read was mapped to a region that is immediately upstream of six consecutive As or of a 10-bp region with at least 60% of As, it was considered as a mis-primed read and removed.The resulting filtered reads were analyzed using a modified version of LAPA 61 that used the default setting, except requiring a replication rate cutoff at 0.75 and can identify polyA site-containing reads mapped to the reverse strand.The change in the polyA site usage was considered significant if the usage difference between two conditions was >10% with adjusted p value < 0.05.PolyA sites were considered cryptic if their usage was ≤5% under the control condition but ≥10% under the TDP-43 knockdown condition and their usage increase was ≥10%.To identify TDP-43 dependent APA events, genes were considered only if they had at least two identified polyA sites and if they had more than two polyA sites, they were filtered for two polyA sites with the two largest usage changes and used for plotting (Table S3).Cryptic polyA sites further include polyA sites that became activated upon TDP-43 knockdown and are located downstream of a gene's annotated 3' end, which is extracted from a curated gene annotation database.Cryptic splicing events detected in RNA-seq data and 3' end-seq data were used to search for coordinated cryptic polyadenylation events.

Calculation of polyA site score
For each polyA site identified in 3' end-seq libraries, a 205 bp sequence centered at site was extracted and used to calculate the polyA site score using Aparent2, which was then converted to log odds ratio.

Evaluation of TMEM106B 3' UTR length in FTLD-TDP brain samples by qRT-PCR
Our study cohort included a total of 83 postmortem cases classified into two main groups: healthy controls (n=27) and Frontotemporal dementia (FTD) cases (n=56).Summary of patient data is included in Table S7.
RNA was extracted from the frontal cortices of the healthy or the FTD patients following the manufacturer's protocol using the RNAeasy Plus Mini Kit (Qiagen) and as previously described 9,11,62 .Up to three cuts of the sample was used for extraction and only the high-quality RNA samples were processed for downstream analysis.RNA concentration was measured by using Nanodrop technologies (Thermo Fisher) and the RNA integrity number (RIN) was evaluated by Agilent 2100 bioanalyzer (Agilent Technologies).Subsequently, 500 ng of the total RNA extracted was reverse transcribed into cDNA using the High-Capacity cDNA Transcription Kit (Applied Biosystems) per the manufacturer's instructions.cDNA samples, in triplicates, with SYBR GreenER qPCR SuperMix (Invitrogen), were further evaluated for the quantitative real-time PCR (qRT-PCR) on a QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems).Relative quantification of the long TMEM106B 3' UTR and total TMEM106B levels was determined using the ΔΔCt method and normalized to two endogenous controls, GAPDH and RPLP0.All the statistical analyses were performed using the GraphPad Prism 10 (GraphPad Software).For comparison of the frontal cortex RNA levels between the healthy and FTD cases, Mann-Whitney test was used.Primers are listed in Table S6.

Construction of luciferase reporters
The short TMEM106B 3' UTR and the long TMEM106B 3' UTR were amplified from human genomic DNA (H1) and then cloned into the pmirGLO Dual-Luciferase Vector (Promega, E1330) using Gibson assembly.The proximal polyA site in the long TMEM106B 3' UTR was then mutated using Gibson assembly.Mutations that

Figure 1 .
Figure 1.Loss of TDP-43 leads to widespread alternative polyadenylation changes.a, APA analysis scheme.Top panel, scheme for sorting neuronal nuclei from frontal cortices of FTD or FTD-ALS patients based on TDP-43 levels for RNA-seq 26 .Bottom panel, workflow of APA analysis using RNA-seq data.b, Loss of TDP-43 is associated with APA changes.APA change (∆PUI by APAlyzer) is plotted on the x-axis and the corresponding statistical significance (the adjusted p value by DEXseq) on the y-axis.Genes with statistically significant APA change (adjusted p < 0.1) are labeled in red for favoring distal polyA sites and in blue for proximal polyA sites.Dashed horizontal line marks the adjusted p value of 0.1.c, NGN2 over-expression mediated differentiation of human stem cells to cortical neurons for shRNA-mediated TDP-43 knockdown (KD).d, Western blot (WB) shows efficient TDP-43 KD after 7 days.p-value was calculated by Student's t test.e, Volcano plot shows widespread APA changes upon TDP-43 KD in iNeurons; thresholds for significant changes are |∆PUI| > 0.10 and adjusted p < 0.05 from APAlyzer.ns (not significant), p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Figure 2 .
Figure 2. High-resolution mapping of TDP-43 dependent alternative polyadenylation using 3' end-seq.a, Top panel, workflow of 3' end-seq.Bottom panel, example of 3' end-seq read coverage, indicative of polyA sites, at the 3' UTR of MRS2.Top track: RNA-seq read coverage; middle track: read coverage of 3' end-seq with peaks corresponding to novel sites marked in green; bottom track: gene structure.b, Top panel, example of TDP-43 regulated APA changes detected by 3' end-seq.The direction of LRFN1 gene is flipped for illustration purpose.Bottom panel, example of premature polyadenylation captured by 3' end-seq.Sashimi plots in the top two tracks illustrate splicing; the pink curved line marks the usage of a cryptic 3' splice site in intron 1 of STMN2.c, Volcano plot shows that 3' end-seq captures widespread APA changes upon TDP-43 knockdown (KD).Thresholds for significant changes are |∆PUI| > 0.10 and adjusted p < 0.05.d, Scatter plot with APA changes, represented by distal polyA site usage, on the x-axis, and RNA

Figure 3 .
Figure 3. Loss of TDP-43 causes alternative polyadenylation that alters protein levels of diseaseassociated genes.

Figure S1 .
Figure S1.Loss of TDP-43 leads to widespread alternative polyadenylation changes.