Identify the specific binding partners of cytosolic TDP-43 WT and TDP-43ΔCR
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Permanent Cell Line Cell
SUBMITTER: 
Ju Gao
LAB HEAD: Xinglong Wang
PROVIDER: PXD027494 | Pride | 2022-05-20
REPOSITORIES: Pride
Publications
Translational regulation in the brain by TDP-43 phase separation.
Gao Ju J Wang Luwen L Ren Xiaojia X Dunn Justin R JR Peters Ariele A Miyagi Masaru M Fujioka Hisashi H Zhao Fangli F Askwith Candice C Liang Jingjing J Wang Xinglong X
The Journal of cell biology 20210824 10
The in vivo physiological function of liquid-liquid phase separation (LLPS) that governs non-membrane-bound structures remains elusive. Among LLPS-prone proteins, TAR DNA-binding protein of 43 kD (TDP-43) is under intense investigation because of its close association with neurological disorders. Here, we generated mice expressing endogenous LLPS-deficient murine TDP-43. LLPS-deficient TDP-43 mice demonstrate impaired neuronal function and behavioral abnormalities specifically related to brain f ...[more]
Similar Datasets
Project description:TDP-43 is an important RNA binding protein. To better understand its binding targets in human neurons, we performed TDP-43 iCLIP on SHSY5Y cells.
2022-02-02 | E-MTAB-11243 | biostudies-arrayexpress
Project description:To clarify the functional properties of Tdp-43, we established the differentially expressed alternative exons in Tdp-43-silenced primary cortical neurons by using exon-sensitive microarray technology. We analyzed total RNA of primary motor neuron infected with lentivirus expressing shRNA against mouse Tdp-43 or control. RNA was harvested 11 days after transfection.
2013-04-18 | E-GEOD-46146 | biostudies-arrayexpress
Project description:In recent times, high throughput screening analyses have broadly defined the RNA cellular targets of TDP-43, a nuclear factor involved in neurodegeneration. A common outcome of all these studies is that changing the expression levels of this protein can alter the expression of several hundred RNAs within cells. What still remains to be clarified is which changes represent direct cellular targets of TDP-43 or just secondary variations due to the general role played by this protein in RNA metabolism. Using a HTS-based splicing junction analysis we have now identified 162 splicing events that are consistent with being directly controlled by TDP-43. Validation of the data, both in neuronal and non-neuronal cell lines demonstrated that TDP-43 substantially alters the levels of isoform expression in four genes potentially important for neuropathology: MADD/IG20, STAG2, FNIP1, and BRD8. Most importantly, for MADD/IG20 and STAG2 these changes could also be confirmed at the protein level. These alterations were also observed in a cellular model that successfully mimics TDP-43 loss of function effects following its aggregation. These novel splicing events may represent potential biomarkers to predict disease onset, progression, and to test the efficacy of novel therapeutic agents to recover TDP-43 functional properties. We have performed an HTS-based splicing junction analysis of a series of stable cell lines that lack TDP-43, overexpress this factor, or express an RNA-binding mutant, in order to find splicing events, potentially associated with neurodegenerativce diseases, regulated by this splicing factor. Samples were analyzed in triplicate from: The following samples were analyzed in triplicate: wild-type HEK-293 cells, siTDP43-treated HEK-293 cells, siTDP43-treated HEK-293 cells overexpressing a flagged-wildtype TDP-43, siTDP43-treated HEK-293 cells overexpressing a RNA-binding deficient mutant.
2015-08-11 | E-GEOD-67195 | biostudies-arrayexpress
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)
2016-08-02 | E-GEOD-85065 | biostudies-arrayexpress
Project description:We identify the RNA targets of TAR DNA-binding protein 43 (TDP-43) from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). We identify 4352 highly enriched RNA targets of TDP-43. We determined that the canonical TDP-43 binding site (TG)n was 55.1-fold enriched in our TDP-43 library. Moreover, our analysis shows there is often an adenine in the middle of the motif, (TG)nTA(TG)m. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to RNA metabolism, neuronal development, and synaptic function. Examination of TDP-43 RNA targets in rat cortical neurons by RIP-seq. Chantelle F. Sephton isolated the TDP-43:RNA complexes and generated the cDNA library for deep sequencing. Email: chantelle.sephton@utsouthwestern.edu Phone: 214-648-4119 Fax: 214-648-1801 ULR: http://www8.utsouthwestern.edu/utsw/cda/dept120915/files/151135.html Organization name: University of Texas Southwestern Medical Center at Dallas Department: Neuroscience Lab: Gang Yu lab Street: 6000 Harry Hines Blvd. City: Dallas State: Texas ZIP: 75390 Country: USA
2010-11-04 | E-GEOD-25032 | biostudies-arrayexpress
Project description:TAR DNA binding protein of 43 kDa (TDP-43) forms aggregates in neurodegenerative diseases, particularly certain forms of frontotemporal dementia and amyotrophic lateral sclerosis. Pathological modifications of TDP-43 include proteolytic fragmentation, phosphorylation, and ubiquitinylation. In this study, we systematically mapped TDP-42 ubiquitination as well as acetylation sites.
2022-04-01 | PXD030170 | Pride
Project description:Mutations in TDP-43 (an RNA binding protein) are known to cause amyotrophic lateral sclerosis (ALS). Previously, our group and several other studies showed that TDP-43 binds to several RNA targets in the mammalian CNS. ALS causing mutations in the C-terminal region of TDP-43 that is involved in splicing regulation may lead to aberrant splicing of several RNA transcripts. The main aim of this study is to identify the effect of an ALS causing mutation on the splicing regulation of previously known targets of TDP-43. Mice overexpressing a mutant human TDP-43 (hTDP-43) gene were obtained from The Jackson Laboratory (strain name - B6;CB-Tg(Prnp-TARDBP*A315T) 95Balo/J; Stock no:010700). Mutant hTDP43 was expressed under the control of a prion protein promoter that drives the expression mainly in the mouse CNS and the male transgenic mice developed symptoms around 12 weeks of age while the female mice develop symptoms approximately 20 weeks of age. Therefore, Tg animals that are 50 days old were considered pre-symptomatic and 100 days old (Tg) animals were considered to be in the post-symptomatic stages of the disease for exon array experiments. Age and sex matched pre and post-symptomatic transgenic animals and their wild type littermates were euthanised by cervical dislocation and tissues (brain and spinal cord) were harvested immediately for total RNA isolation experiments (n=3 per group). 200 ng of total RNA from transgenic TDP-43 mice and wild type animals was converted to cDNA and then amplified using the Applause WT-Amp Plus ST kit (NuGEN). Amplified cDNA was then fragmented and biotin labeled using the Encore Biotin Module kit (NuGEN) according to the manufacturer's instruction manual. Labelled cDNA was then hybridised onto Affymetrix GeneChip Exon 1.0 ST arrays in a hybridisation oven at 45 degrees C for 20 hours at 60rpm. After hybridization, washing and staining of the arrays were carried out using Affymetrix fluidics station 450 followed by scanning using GeneChip scanner. GeneChip Command Console Software (AGCC) controlled both the fluidics station and the scanner. CEL files generated by AGCC were uploaded onto Partek software and the data analysis was carried out using the exon array analysis workflow. Exon 1.0 ST arrays contain many more probes, which are classified into three major types based on their source. They are Core, extended and full probe set annotation. Core annotation refers the probe sets that are the most reliable of the three and is derived based on evidences from Refseq and GenBank. Extended annotations refer to probe sets that are generated based on EST sequences, ENSEMBL gene collections and other databases including those used for generating core probe sets. The full annotation refers to probe sets that are purely based on computational predictions. Core probe set annotation, unlike the extended or full annotation excludes the speculative probes reducing the incidence of false positives and was employed for exon array analysis. Alt-splice Anova, a statistical tool available in Partek was used for identifying novel alternative splicing events.
2014-02-10 | E-MTAB-1487 | biostudies-arrayexpress
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:TDP-43 is a nuclear protein involved in pivotal processes, extensively studied for its implication in neurodegenerative disorders. TDP-43 cytosolic inclusions are a common neuropathologic hallmark in amyotrophic lateral sclerosis (ALS) and related diseases, and it is now established that TDP-43 misfolding and aggregation play a key role in their etiopathology. TDP-43 neurotoxic mechanisms are not yet clarified, but the identification of proteins able to modulate TDP-43-mediated damage may be promising therapeutic targets for TDP-43 proteinopathies. Here we show by the use of refined yeast models that the nucleolar protein nucleolin (NCL) acts as a potent suppressor of TDP-43 toxicity, restoring cell viability. We provide evidence that NCL co-expression is able to alleviate TDP-43-induced damage also in human cells, further supporting its beneficial effects in a more consistent pathophysiological context. Presented data suggest that NCL could promote TDP-43 nuclear retention, reducing the formation of toxic cytosolic TDP-43 aggregates.
2021-03-26 | PXD022432 | Pride
Project description:In total 3 mice brain tissues were used for this experiment. Tissue from each mouse brain was divided for immunoprecipitation with 5ug of either rabbit anti-TDP-43 antibody (Abcam) or normal rabbit IgG (Sigma) .The antibodies were first incubated with the lysate overnight at 4 degrees C, after which 50 uL of protein G Dynabeads(tm) (Invitrogen(tm)) added and the solution incubated for 1 hour at 4 degrees C with rotation. Following several washing with washing buffer (Invitrogen(tm)), the protein-RNA complex was eluted from 20 uL of bead-protein complex using 10 uL of elution buffer (Invitrogen(tm)) and seperated on a 10% NuPage(tm) Bis-Tris gel (Invitrogen(tm)). RNA was isolated from the remaining 30 uL of protein-bead complex using TRIzol reagent (Invitrogen) followed by DNaseI treatment (Ambion). The TDP-43 immunoprecipitated RNA was converted to cDNA, fragmented and biotin labelled using WT cDNA synthesis & amplification kit and Terminal Labeling Kit (Affymetrix(tm)) for Affymetrix(tm) GeneChip(tm) Mouse Gene 1.0 ST and 3'-IVT expression analysis kit (Affymetrix(tm)) for GeneChip(tm) Mouse 430_2 arrays according to the standard protocol. Labelled RNA was hybridised to arrays in a hybridization oven (Affymetrix(tm)) at 45 degrees C rotating at 60 rpm for 17 hours and scanned using the Affymetrix(tm) GeneChip Scanner. Successful hybridisation to the microarray was checked using Expression Console software (Affymetrix(tm)) and the data (.CEL files) transferred to Partek Genomics Suite for statistical analysis. TDP-43 and IgG immunoprecipitated RNA samples were each hybridised to 3 GeneChip Mouse Gene 1.0 ST and 3 GeneChip Mouse 430 (n = 6 for each group).
2012-02-06 | E-MEXP-3500 | biostudies-arrayexpress