3'-end targeted sequencing of 6 control and 6 TDP-43 KD samples in HEK293
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB14143 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR1421770.fastq.gz | Fastqsanger.gz | |||
| ERR1421771.fastq.gz | Fastqsanger.gz | |||
| ERR1421772.fastq.gz | Fastqsanger.gz | |||
| ERR1421773.fastq.gz | Fastqsanger.gz | |||
| ERR1421774.fastq.gz | Fastqsanger.gz |
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Project description:This project studies TDP43, which is an RNA binding protein implicated in Motor Neuron Disease. As an RNA-binding protein, TDP43 is known to influences poly-adenylation site choice. For this project, we have inserted a single copy of the GFP-tagged TDP43 gene into the FLPIn Locus of HEk293 cells. We use these Hek293 FLipIn lines to instigate the effect of different deletion and mutation constructs of TDP-43 in their ability to rescue the depletion (siRNA) of the endogenous TDP-43 protein. We are comparing siRNA mediated KD in triplicates for each of the 7 cell lines to the Dox-induced rescues in triplicates. We are using a customised Lexogen Quantseq 3’ end sequencing method that allows us to multiplex cDNAs straight after the reverse transcription. The samples were pooled into barcoded sub-groups, each group will have the Lexogen barcode (i7 indices) in addition.
2021-07-21 | E-MTAB-9410 | biostudies-arrayexpress
Project description:HEK293 FlpIn T-Rex cells were maintained in Dulbeccos Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), supplemented with 3 g/ml blasticidine and 50 g/ml zeocin. For the siRNA-induced knockdown of TDP-43, 20 nM of TDP-43 stealth siRNA was mixed with 10 L of RNAiMAX following the manufacturers reverse transfection protocol and added to a 10 cm dish of HEK FlpIn cells. For the non-targeting siRNA also 20 nM was used, to distinguish off-target effects from biologically relevant ones. After the first 24 hrs of transfection, the medium was replaced with DMEM with 10% FBS and after an additional 24 hrs, the cells were collected for analysis. RNA was extracted using the Direct-zol RNA kit and an in-column DNase digestion step was performed at room temperature for 15 minutes. The poly(A)seq libraries for samples, that were transfected with either non-targeting siRNA or TARDBP siRNA, were generated using the reverse QuantSeq 3mRNA-Seq kit. Libraries were prepared from 500 ng of total RNA. In the protocol one fragment per transcript was generated, which resulted in extremely accurate gene expression values. For the initial step of this kit, oligodT priming, including Illumina-compatible linker sequences, was carried out. The second strand synthesis was followed by purification with magnetic beads. Barcodes were introduced during the PCR amplification step as standard external barcodes. Single-end sequencing (60 nts) was performed on a Illumina GA-2 with a Rapid Run flow-cell. This kit makes use of a custom sequencing primer that anneals to a linker sequence previously introduced in the oligo(dT) priming step for reverse transcription.
2017-05-01 | E-MTAB-4732 | biostudies-arrayexpress
Project description:Nuclear depletion and cytoplasmic aggregation of the RNA-binding protein TDP-43 is the hallmark of ALS, occurring in over 97% of cases. A key consequence of TDP-43 nuclear loss is the de-repression of cryptic exons. Whilst TDP-43 regulated cryptic splicing is increasingly well catalogued, cryptic alternative polyadenylation (APA) events, which define the 3’ end of last exons, have been largely overlooked, especially when not associated with novel upstream splice junctions. We developed a bioinformatic pipeline to reliably identify distinct APA event types: alternative last exons (ALE), 3’UTR extensions (3’Ext) and ‘ composite ’ intronic polyadenylation (IPA) events. We identified novel neuronal cryptic APA sites induced by TDP-43 loss of function by systematically applying our pipeline to a compendium of publicly available and in house datasets. We find that TDP-43 binding sites and target motifs are enriched at these cryptic events and that TDP-43 can have both repressive and enhancing action on APA. Importantly, all categories of cryptic APA can also be identified in ALS and FTD post mortem brain regions with TDP-43 proteinopathy underlining their potential disease relevance. RNA-seq and Ribo-seq analyses indicate that distinct cryptic APA categories have different downstream effects on transcript and translation. Intriguingly, cryptic 3’Exts occur in multiple transcription factors, such as ELK1 , SIX3, and TLX1, and lead to an increase in RNA stability. 3’Exts are localised to the cytoplasm and neurites and translated leading to an increase in wild-type protein levels. In summary, we demonstrate that TDP-43 nuclear depletion induces a novel category of cryptic RNA processing events and we expand the palette of TDP-43 loss consequences by showing this can also lead to an increase in normal protein translation.
2025-05-30 | GSE296712 | GEO
Project description:Nuclear depletion and cytoplasmic aggregation of the RNA-binding protein TDP-43 is the hallmark of ALS, occurring in over 97% of cases. A key consequence of TDP-43 nuclear loss is the de-repression of cryptic exons. Whilst TDP-43 regulated cryptic splicing is increasingly well catalogued, cryptic alternative polyadenylation (APA) events, which define the 3’ end of last exons, have been largely overlooked, especially when not associated with novel upstream splice junctions. We developed a bioinformatic pipeline to reliably identify distinct APA event types: alternative last exons (ALE), 3’UTR extensions (3’Ext) and ‘ composite ’ intronic polyadenylation (IPA) events. We identified novel neuronal cryptic APA sites induced by TDP-43 loss of function by systematically applying our pipeline to a compendium of publicly available and in house datasets. We find that TDP-43 binding sites and target motifs are enriched at these cryptic events and that TDP-43 can have both repressive and enhancing action on APA. Importantly, all categories of cryptic APA can also be identified in ALS and FTD post mortem brain regions with TDP-43 proteinopathy underlining their potential disease relevance. RNA-seq and Ribo-seq analyses indicate that distinct cryptic APA categories have different downstream effects on transcript and translation. Intriguingly, cryptic 3’Exts occur in multiple transcription factors, such as ELK1 , SIX3, and TLX1, and lead to an increase in RNA stability. 3’Exts are localised to the cytoplasm and neurites and translated leading to an increase in wild-type protein levels. In summary, we demonstrate that TDP-43 nuclear depletion induces a novel category of cryptic RNA processing events and we expand the palette of TDP-43 loss consequences by showing this can also lead to an increase in normal protein translation.
2025-05-30 | GSE296711 | GEO
Project description:To investigate how translation changed as a function of TDP-43 CR loss, mass spectrometry (MS) based approach was performed to monitor the dynamics of TDP-43-associated protein complexes in response to CR deletion using TDP-43 knockout HEK293 cells expressing strep tagged wild type TDP-43 (TDP-43WT) or TDP-43ΔCR mutant.
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Project description:A stable HEK293 FlpIn T-Rex cells expressing TDP-43 with an N-terminal eGFP-tag was generated that allowed inducible physiological expression of the protein (Ling et al. 2010). Duplicate iCLIP experiments were performed using an antibody targeting eGFP (Abcam ab290). Crosslinked RNA-protein complexes were isolated by immuno-precipitation and cDNAs were generated to allow preparation of Illumina compatible DNA libraries as described in Huppertz et al. (2014).
2017-05-01 | E-MTAB-4733 | biostudies-arrayexpress
Project description:Cytoplasmic aggregation and nuclear depletion of TDP-43 are hallmarks of several age-related neurodegenerative disorders. Yet, recapitulating both features in cellular systems has been a major challenge. Here, we produced amyloid-like fibrils from the recombinant low-complexity-domain of TDP-43, and demonstrate that sonicated fibrils trigger TDP-43 pathology in human cell lines and iPSC-derived neurons. Fibril-induced cytoplasmic TDP-43 inclusions acquire distinct biophysical properties, recapitulate pathological hallmarks such as phosphorylation, ubiquitin- and p62-accumulation, and recruit nuclear endogenous TDP-43 leading to nuclear-loss-of-function-driven disease-specific cryptic splicing defects. Cytoplasmic TDP-43 aggregates exhibit, with time, distinct heterogeneous morphologies as in patients including compacted, filamentous or fragmented, through the activation of cellular protein clearance pathways. Cell-specific progressive toxicity is provoked by seeded TDP-43 pathology in human neurons. These findings identify templated aggregation of TDP-43 as a key mechanism driving both cytoplasmic gain- and nuclear-loss-of-function, offering a valuable approach to identify modifiers of sporadic TDP-43 proteinopathies.
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Project description:MicroRNAs (miRNAs) play important roles in a wide range of cellular processes. Aberrant regulation of miRNA genes contributes to human diseases, including cancer. The TAR DNA binding protein 43 (TDP-43), a DNA/RNA binding protein associated with neurodegeneration, is involved in miRNA biogenesis. Here, we systematically examined miRNAs whose expression levels are regulated by TDP-43 using RNA-Seq coupled with siRNA-mediated knockdown approach. TDP-43 knocking down affected the expression of a number of miRNAs. Alterations in isomiR patterns and miRNA arm selection after TDP-43 knockdown suggest a role of TDP-43 in miRNA editing. We examined correlation of selected TDP-43 associated miRNAs and their candidate target genes in human cancers. Our data reveal highly complex roles of TDP-43 in regulating different miRNAs and their target genes. Our results suggest that TDP-43 may promote migration of lung cancer cells by regulating miR-423-3p expression. On the other hand, TDP-43 increases miR-500a-3p expression and binds to the mature miR-500a-3p sequence. Low expression of miR-500a-3p was associated with poor survival of lung cancer patients, suggesting that TDP-43 may have a suppressive role in cancer by regulating miR-500a-3p. Our experiments reveal that cancer-associated genes LIF and PAPPA may be targets of miR-500a-3p. Together with other studies, our work suggests that TDP-43-regulated miRNAs may play multi-facet roles in the pathogenesis of cancer. small RNA seq in SH-SY-5Y, SNB-19 and HT22 (TDP-43 siRNA VS Control siRNA)
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