Biology subjects

Onodera, O.

Publications and source records attributed to Onodera, O..

7 recordsLinked to original sources

bioRxiv2026

Exitron-type splicing of intrinsically disordered regions recurs across jawed vertebrates despite sequence divergence: donor-proximal cis control in TARDBP

Intrinsically disordered regions (IDRs) mediate molecular interactions but can also promote aberrant self-association. Using donors embedded within IDR-coding sequences, exitron-type splicing can remove these sequences, giving them both protein-coding and splice-regulatory roles. Yet IDR amino-acid sequences often diverge rapidly, raising the question of whether and how their coding sequences support IDR-removing splicing across species. Using RNA-seq data from jawed vertebrates, we identified events predicted to remove most of an IDR while preserving annotated structured regions. Across species, donor-flanking nucleotide identity exceeded expectations from synonymous alternatives encoding the same amino-acid sequence. TARDBP, which encodes TDP-43, provided a model for examining how coding sequences both encode IDRs and regulate exitron-type splicing. Sequence-divergent vertebrate TARDBP terminal regions supported this splicing in human cells. In teleosts, TARDBP ohnologs (tardbpa and tardbpb) encoded compositionally similar disordered tails but differed markedly in IDR-removing exitron-type splicing. Reciprocal donor-proximal substitutions in zebrafish minigenes redirected donor choice and altered the balance between IDR-retaining and IDR-removing transcripts. In corresponding reporters, these substitutions altered protein-product balance and cytoplasmic puncta formation. These findings show that IDR-removing exitron-type splicing can recur despite sequence divergence and that donor-proximal TARDBP sequences both encode IDR segments and regulate IDR inclusion, linking RNA processing to protein behavior.

molecular biology↗

bioRxiv2025

Export-biased, 3'UTR-preserving TDP-43 model links nuclear loss to cytoplasmic aggregation in ALS/FTLD

Nuclear depletion and cytoplasmic mislocalization of TDP-43 are central pathological features of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 protein levels are normally maintained by autoregulation through its native 3' untranslated region (3' UTR). It remains untested whether this feedback is still protective once the perturbation is chronic, and whether the resulting rise in TARDBP transcripts restores the functional nuclear pool or is instead diverted into non-functional species. To address this, we engineered full-length human TDP-43 carrying an N-terminal nuclear export signal (NES) while leaving the native 3' UTR autoregulatory module intact. Expression was titrated so that whole-cell RIPA-soluble exogenous TDP-43 remained [≤]30% of endogenous levels. In HEK293T cells, export bias drove TDP-43 into the cytoplasm and produced detergent-insoluble species. In differentiated SH-SY5Y cells, nuclear splicing defects and autoregulatory changes scaled with export-biased load, and detergent-insoluble accumulation was already present within the same low-load range. Human iPSC-derived neurons showed a comparable cytoplasmic shift, discrete TDP-43-positive foci, and TDP-43-dependent splicing defects. When endogenous TDP-43 was selectively depleted under transgene induction, 3' UTR-coupled autorepression was weakened and transgene-derived TARDBP transcripts rose; however, the additional exogenous TDP-43 did not expand the soluble, splice-competent pool but partitioned into insoluble fractions. Splicing defects in differentiated SH-SY5Y cells persisted even when soluble NES-TDP-43 reached endogenous-equivalent levels. We therefore propose that under native 3' UTR control, sustained cytoplasmic bias uncouples compensatory TARDBP upregulation from recovery of the functional nuclear pool; the extra output is diverted into insoluble, fragmented species rather than restoring nuclear function.

molecular biology↗

bioRxiv2024

ASO-enhancement of TARDBP exitron splicing mitigates TDP-43 proteinopathies

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are fatal neurodegenerative diseases characterized by pathological aggregation and nuclear functional loss of TDP-431,2. Current therapies inadequately address this core pathology3,4, necessitating innovative approaches that target aggregation while preserving TDP-43s essential functions. Here we demonstrate that enhancing the splicing of the TARDBP exitron--a cryptic intron encoding the aggregation-prone intrinsically disordered region (IDR) of TDP-435,6-- effectively mitigates TDP-43 pathology. This exitron splicing event, directly regulated by nuclear TDP-437-9, suppresses the expression of IDR-containing TDP-43 isoforms and generates IDR-spliced-out TDP-43 isoforms7,9,10 (which we term "IDRsTDP"). Our findings reveal that IDRsTDP, known to heterodimerize with full-length TDP-4310, inhibits TDP-43 aggregation by suppressing IDR-mediated clustering and enhances TDP-43 clearance via chaperone-mediated autophagy. In disease states, however, impaired nuclear TDP-43 function disrupts exitron splicing, leading to increased levels of IDR-containing TDP-439,11 and reduced levels of IDRsTDP, exacerbating aggregation and nuclear dysfunction6,12-17. By identifying HNRNPA1 and HNRNPC as key repressors of TARDBP exitron splicing, we designed antisense oligonucleotides (ASOs) to block their binding and restore splicing. These ASOs suppressed TDP-43 pathology and neurodegeneration in both neuronal cell models with impaired nuclear transport and a mouse model of proteasome dysfunction-induced TDP-43 proteinopathy. Our strategy, by rescuing the impaired autoregulatory pathway, inhibits the pathological cycle of TDP-43 aggregation and nuclear dysfunction, offering a promising avenue for treating these currently intractable neurodegenerative diseases.

neuroscience↗

bioRxiv2022

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

neuroscience↗

bioRxiv2022

Accuracy of a machine learning method based on structural and locational information from AlphaFold2 for predicting the pathogenicity of TARDBP and FUS gene variants in ALS

BackgroundIn the sporadic form of amyotrophic lateral sclerosis (ALS), the pathogenicity of rare variants in the causative genes characterizing the familial form remains largely unknown. To predict the pathogenicity of such variants, in silico analysis is commonly used. In some cases of ALS, the gene mutations are concentrated in specific regions, and the resulting alterations in protein structure are thought to significantly affect pathogenicity. However, existing methods have not taken this issue into account. To address this, we have developed a technique termed MOVA (method for evaluating the pathogenicity of missense variants using AlphaFold2), which applies positional information for structural variants predicted by AlphaFold2. Here we examined the utility of MOVA for analysis of several causative genes of ALS. MethodsWe analyzed variants of six ALS-related genes (TARDBP, FUS, SETX, TBK1, OPTN, and SOD1) and classified them as pathogenic or neutral. For each gene, the features of the variants, including their positions in the 3D structure predicted by AlphaFold2, were entered into a random forest algorithm and evaluated by leave-one-out cross-validation. We compared how accurately MOVA was able to classify the pathogenic and neutral mutation variants. ResultsMOVA yielded useful results (AUC [≥]0.70 for 3 (TARDBP 0.755, FUS 0.844, and SOD1 0.787) of the 6 genes) and was particularly useful for genes where pathogenic mutations were concentrated at specific sites (TARDBP, FUS). ConclusionsMOVA is useful for predicting the virulence of rare variants of ALS-causing genes in which mutations are concentrated at specific structural sites.

bioinformatics↗

bioRxiv2021

Age-related demethylation of the TDP-43 autoregulatory region in the human motor cortex

In amyotrophic lateral sclerosis (ALS), TAR DNA-binding protein 43 (TDP-43), which is encoded by TARDBP, forms aggregates in the motor cortex. This aggregate formation may be triggered by an increase in the TDP-43 level with aging. However, the amount of TDP-43 is autoregulated by alternative splicing of the TARDBP 3UTR, and how this autoregulation is affected by aging remains to be elucidated. We found that DNA demethylation in the autoregulatory region in the TARDBP 3'UTR reduced alternative splicing and increased TARDBP mRNA expression. Furthermore, in the human motor cortex, we found that this region was demethylated with aging, resulting in increased expression of TARDBP mRNA. The acceleration of DNA demethylation in the motor cortex was associated with the age of ALS onset. In summary, the dysregulation of TDP-43 autoregulation by age-related DNA demethylation in the motor cortex may explain the contribution of aging and motor system selectivity in ALS.

molecular biology↗

bioRxiv2019

Highly efficient direct conversion of human monocytes into neuronal cells using a small molecule combination

Previous studies reported that human fibroblasts and astrocytes were successfully converted into neuronal cells by small molecules without introducing ectopic transgenes. Induced neuronal cells--reprogrammed directly from dermal fibroblasts or brain astrocytes--were obtained from some donors; however, the clinical applications of this approach would be limited because it requires an invasive biopsy to harvest enough cells for derivation. Here, we report that adult human peripheral blood monocytes may be directly converted into neuron-like cells using only a combination of small molecules without transgene integration. This method enables neuronal cell generation from TUJ1-positive cells after 3 days of induction (at over 80% conversion efficacy). These cells presented neuronal morphologies and markers, suggesting that terminally differentiated human cells may be efficiently transdifferentiated into a distantly related lineage. Overall, our study provides a strategy to develop neuronal cells directly from human adult peripheral blood monocytes using a generate transgene-free, chemical-only approach.

cell biology↗