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RNA pull down assay of GST-ZFP36L1 (wildtype and mutant)

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE136179 | GEO | 2020/10/02

REPOSITORIES: GEO

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Silencing of ZFP36L1 using siRNA

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

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RNA pull down assay of GST-ZFP36L1 (wildtype and mutant)

Project description:RNA pull down assay of GST-ZFP36L1 (wildtype and mutant)

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The effect of ZFP36L1 deficiency on gene expression in the U2OS osteosarcoma cancer cell line

Project description:Post-transcriptional regulation of gene expression by RNA binding proteins (RBPs), one of the major classes of proteins encoded by the human genome, is well established. ZFP36L1 and its protein family members, ZFP36 and ZFP36L2, function as RBPs that primarily regulate gene expression at the post-transcriptional level by binding to adenine uridine (AU) rich elements (AREs) in the 3′ untranslated region (3′UTR) of certain mRNAs and mediating ARE-dependent mRNA decay. In this study, using CRISPR/Cas9 ZFP36L1 gene-editing, we generated ZFP36L1 deficient U2OS cellular models. The aim of the RNASeq experiment was to compare the gene expression profile in wild-type U2OS cells compared to ZFP36L1 deficient U2OS cells. Loss of functional ZFP36L1 leads to widespread changes in gene expression that are associated with dysregulation of KRAS signalling.

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Pre-T cells from control and Zfp36l1, Zfp36l2 double conditional knockout mice. [iCLIP]

Project description:Purpose: We aimed to identify the targets of the RNA binding protein ZFP36L1 in thymoctes. Methods: Total naÃ¯ve thymocytes from control or DCKO mice were treated with UV light to crosslink RNA and proteins, then RNA-protein complexes were pulled down with anti-ZFP36L1. RNA was extracted and used to make cDNS libraries that were then sequenced by MiSeq 150bp single-end read (Sample 1) and HiSeq2500 RapidRun 50bp single-end read (sample 2, 3). Results: Sample demultiplexing was performed by identification of the 3 known bases of the 7 bases barcode introduced in the 5â end of the read by the RCLIP primer. The remaining four random bases were used to remove PCR duplicate reads. Reads were trimmed to remove any adaptor sequence and barcodes before mapping reads to genome GRCm38 using Bowtie. After read mapping, the single-nucleotide at position -1 was annotated as unique ZFP36L1 crosslink site. Identification of highly significant ZFP36L1 binding sites was performed using iCount to assign a FDR to each crosslink site. Conclusions: We identified ZFP36L1 binding sites in 8675 thymocyte mRNAs. Individual nucleotide resolution cross linking immunoprecipitation (iCLIP) of ZFP36L1-bound RNAs in total naive thymocytes from C57BL/6 mice.

2016-09-05 | E-GEOD-79178 | biostudies-arrayexpress

Late pre-B cell transcriptomes from Zfp36l1 Zfp36l2 double knockout mice [iCLIP]

Project description:Purpose: We aimed to identify the targets of the RNA binding protein ZFP36L1 in B cells. Methods: Mature B cells were stimulated with LPS, IL-4 and IL-5 for 48 hours to induce ZFP36L1 expression. Cells were treated with UV light to crosslink RNA and proteins then RNA-protein complexes were pulled down with anti-ZFP36L1. RNA was extracted and used to make cDNS libraries that were then sequenced using Illumina’s HiSeq2000 (100 bp single end sequencing). Results: Sample demultiplexing was performed by identification of the 3 known bases of the 7 bases barcode introduced in the 5’ end of the read by the RCLIP primer. The remaining four random bases were used to remove PCR duplicate reads. Reads were trimmed to remove any adaptor sequence and barcodes before mapping reads to genome mm10 using Bowtie. After read mapping, the single-nucleotide at position -1 was annotated as unique ZFP36L1 crosslink site. Identification of highly significant ZFP36L1 binding sites was performed using iCount to assign a FDR to each crosslink site Conclusions: We identified ZFP36L1 binding sites in 1361 B cell mRNAs.

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High-throughput sequencing of Brf1 (Zfp36l1) and Brf2 (Zfp36l2) targets

Project description:We conducted RNA/RNA binding protein immunoprecipitation (RIP) followed by high throughput sequencing to identify mRNA targets of Brf1 (Zfp36l1) and Brf2 (Zfp36l2) in mouse embryonic stem cells. We collected over 190 million sequencing reads and identified many highly enriched protein coding mRNAs. Gene ontology analysis of enriched genes reveals that transcription factors and intercellular signaling proteins account for a large fraction of the targets, many of which are important for pluripotency and differentiation. Identification of Brf1 and Brf2 mRNA targets

2014-04-30 | E-GEOD-40104 | biostudies-arrayexpress

Roles of Zfp36l1 and Zfp36l1 in retinal development

Project description:We report our study of the function of two members of the TTP (tristetraprolin) mRNA binding protein family, Zfp36l1 and Zfp36l2, in retinal development. We found that Zfp36l1 and Zfp36l2 were expressed in retinal progenitor cells during development and Müller glial cells and photoreceptors in the mature retina. Our analysis of the mutant retinas showed that, whereas the single knockout retinas appeared largely normal, the double knockout (DKO) retina manifested decreased RPC proliferation and increased differentiation of multiple retinal cell types. RNA-seq analysis not only confirmed the imbalance of proliferation and differentiation in the DKO retina but also revealed Zfp36l1 and Zfp36l2 interact with multiple signaling pathways including the sonic hedgehog pathway and the Notch pathway, to regulate this process.

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