RNA pull down assay of GST-ZFP36L1 (wildtype and mutant)
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA561512 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved Adenylate-Uridylate-rich Elements (AREs) located in 3' untranslated regions (UTRs) to mediate RNA decay. We hypothesized that ZFP36L1 is a negative regulator of a post-transcriptional hub involved in the RNA half-life regulation of cancer-related transcripts. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo; whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wildtype and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells ± tet-on ZFP36L1, was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1, including HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3' UTRs of these targets for RNA degradation, thus suppressing their expression. Collectively, our findings reveal an indispensable role of ZFP36L1 as a post-transcriptional safeguard against aberrant hypoxic signaling and abnormal cell cycle progression.
2020-10-02 | GSE136179 | GEO
Project description:ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved Adenylate-Uridylate-rich Elements (AREs) located in 3' untranslated regions (UTRs) to mediate RNA decay. We hypothesized that ZFP36L1 is a negative regulator of a post-transcriptional hub involved in the RNA half-life regulation of cancer-related transcripts. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo; whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wildtype and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells ± tet-on ZFP36L1, was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1, including HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3' UTRs of these targets for RNA degradation, thus suppressing their expression. Collectively, our findings reveal an indispensable role of ZFP36L1 as a post-transcriptional safeguard against aberrant hypoxic signaling and abnormal cell cycle progression.
2020-10-02 | GSE136180 | GEO
Project description:ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved Adenylate-Uridylate-rich Elements (AREs) located in 3' untranslated regions (UTRs) to mediate RNA decay. We hypothesized that ZFP36L1 is a negative regulator of a post-transcriptional hub involved in the RNA half-life regulation of cancer-related transcripts. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo; whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wildtype and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells ± tet-on ZFP36L1, was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1, including HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3' UTRs of these targets for RNA degradation, thus suppressing their expression. Collectively, our findings reveal an indispensable role of ZFP36L1 as a post-transcriptional safeguard against aberrant hypoxic signaling and abnormal cell cycle progression.
2020-10-02 | GSE136181 | GEO
Project description:RNA isolated from MLE12 cells was subjected to a pull down assay with biotinilated miR-154 to enrich for miR-154 target mRNAs
2020-05-21 | GSE141292 | GEO
Project description:Identification of proteins binding to the promoter of hrpR gene in Pseudomonas syringae using DNA pull-down & MS assay
2021-03-23 | PXD023722 | Pride
Project description:RNA pull-down assay.<br>For the recombinant protein pull-down assays, 50 M-5g of recombinant His-tag TcRBP40 protein were bound to 100 uL of Ni-NTA resin (Qiagen) overnight at 4M-0C. 100 M-5g of total RNA from epimastigotes were incubated with the bound protein in 500 M-5l EMSA buffer at 4M-0C for 2 h, in the presence of Heparine and Spermidine as competitors. Bounded and supernatant samples were separated. The bound sample was washed with the same buffer three times, soft-mixing for 10 min each. After washing, RNA present in the bound and supernatantM- fractions were purified.<br><br>RNA purification and amplification:<br><br>RNA was extracted using the RNeasy mini kit (Qiagen). Linearly amplified RNA (aRNA) was generated with the MessageAmpM-^YII aRNA Amplification kit (Ambion), according to the manufacturerM-^Rs manual.<br><br>Microarray analysis:<br>The microarray was constructed with 70-mer oligonucleotides. Due to the hybrid and repetitive nature of the sequenced T. cruzi strain, all coding regions (CDS) identified in the genome (version 3) were retrieved and clustered by the BLASTClust program, using parameters of 40% coverage and 75% identity. For probe design, it was used ArrayOligoSelector software (v. 3.8.1), with a parameter of 50% G+C content. Was obtained 10,359 probes for the longest T. cruzi CDS of each cluster, 393 probes corresponded to the genes of an external group (Cryptosporidium hominis) and 64 spots contained only spotting solution (SSC 3x), given 10,816 spots in total. These oligonucleotides were spotted from a 50 M-5M solution onto poly-L-lysine coated slides and cross-linked with 600 mJ UV. Each probe corresponding to the T. cruzi genes was identified according to the T. cruzi Genome Consortium annotation (www.genedb.org). We compared bound and unbound mRNA, extracted from two independent pull-down assays, in a dye-swap design including four slides. <br>Microarray images were analyzed by Spot software (Spot). The Limma package (Smyth GK, 2004) was used for background correction by the normexp method, intra-slide normalization by the printtiploess method and inter-slide normalization by the quantile method. The results for the two intra-slide probe replicates were then averaged. The pull-down results were averaged, and probes displaying more than a two-fold difference between the bound and unbound fractions were selected, at FDR 1%.
2012-06-30 | E-MEXP-3057 | biostudies-arrayexpress
Project description:In vitro pull down assay was performed using recombinant GST and GST fusion SOX2 protein to precipitate total RNA from urothelial carcinoma cell line BFTC905 Unbound RNA from both reactions and matrix-associated RNA from SOX2 pull down reaction was extracted using TRIZOL reagent
2011-10-26 | GSE33207 | GEO
Project description:We aimed to discover trans-acting RNA molecules involved in mRNA 3’ processing. We reasoned that, if there exist such functional RNAs, they must directly associate with the key machinery responsible for mRNA 3’ processing. Therefore, it would be of great value to comprehensively identify RNAs interacting with pre-mRNA 3’ processing complex. To this goal, we took advantage of previously well-characterized system combined with high-throughput sequencing to investigate the target RNAs at the transcriptomic level. Briefly, we used polyA site (PAS) RNA substrates, SV40 late (SVL), and corresponding control RNAs with point mutation (U to C) at the highly conserved cis-element AAUAAA. RNA substrates were first biotinylated at the 3’ end, and then bound to the streptavidin magnetic beads. After incubation with Hela Nuclear Extract (NE) under polyadenylation condition, the two wild type RNA substrates could specifically recruit mRNA 3’ processing factors in NE for complex assembly. The purification of the protein complex and its interacting RNAs were performed using biotin-streptavidin pull-down. We extracted RNAs from the pull-down sample, prepared the strand-specific RNA-Seq libraries and submitted them for deep-sequencing.
2017-07-01 | GSE98218 | GEO
Project description:High-throughput sequencing analysis of transcriptome from LPS-activated B cells from Lymph node of Zfp36l1(fl/fl) and Zfp36l1(fl/fl) x Cd79a(Cre/+) mice
2020-11-20 | GSE146073 | GEO