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Large-scale tethered function assays identify factors that regulate mRNA stability and translation (eCLIP)

ABSTRACT: Large-scale tethered function assays identify factors that regulate mRNA stability and translation (eCLIP)

PROVIDER: PRJNA482135 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11683600.fastq.gz	Fastqsanger.gz
	SRR11683601.fastq.gz	Fastqsanger.gz
	SRR11683602.fastq.gz	Fastqsanger.gz
	SRR11683603.fastq.gz	Fastqsanger.gz
	SRR11683604.fastq.gz	Fastqsanger.gz

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Large-scale tethered function assays identify factors that regulate mRNA stability and translation (eCLIP)

Project description:Enhanced UV crosslinking and immunoprecipitation (eCLIP) identifies thousands of endogenous mRNA targets for these prioritized RBPs that respond to protein level changes reminiscent of their effects in tethered functional assay.

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Large-scale tethered function assays identify factors that regulate mRNA stability and translation

Project description:Large-scale tethered function assays identify factors that regulate mRNA stability and translation

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Large-scale tethered function assays identify factors that regulate mRNA stability and translation (polysome profiling)

Project description:Large-scale tethered function assays identify factors that regulate mRNA stability and translation (polysome profiling)

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Large-scale tethered function assays identify factors that regulate mRNA stability and translation (HEK293T_RNA-seq)

Project description:Large-scale tethered function assays identify factors that regulate mRNA stability and translation (HEK293T_RNA-seq)

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Large-scale tethered function assays identify factors that regulate mRNA stability and translation

Project description:This SuperSeries is composed of the SubSeries listed below.

2020-08-18 | GSE117294 | GEO

Large-scale tethered function assays identify factors that regulate mRNA stability and translation.

Project description:The molecular functions of the majority of RNA-binding proteins (RBPs) remain unclear, highlighting a major bottleneck to a full understanding of gene expression regulation. Here, we develop a plasmid resource of 690 human RBPs that we subject to luciferase-based 3'-untranslated-region tethered function assays to pinpoint RBPs that regulate RNA stability or translation. Enhanced UV-cross-linking and immunoprecipitation of these RBPs identifies thousands of endogenous mRNA targets that respond to changes in RBP level, recapitulating effects observed in tethered function assays. Among these RBPs, the ubiquitin-associated protein 2-like (UBAP2L) protein interacts with RNA via its RGG domain and cross-links to mRNA and rRNA. Fusion of UBAP2L to RNA-targeting CRISPR-Cas9 demonstrates programmable translational enhancement. Polysome profiling indicates that UBAP2L promotes translation of target mRNAs, particularly global regulators of translation. Our tethering survey allows rapid assignment of the molecular activity of proteins, such as UBAP2L, to specific steps of mRNA metabolism.

| S-EPMC8221285 | biostudies-literature

Large-scale tethered function assays identify factors that regulate mRNA stability and translation (polysome profiling)

Project description:Performed polysome profiling in cell lysates from two independent UBAP2L knockout clonal isolates and from two control samples.

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Large-scale tethered function assays identify factors that regulate mRNA stability and translation (HEK293T_RNA-seq)

Project description:we either depleted RBPs by lentiviral transduction of short-hairpin RNAs (shRNAs) or ectopically expressed the open reading frame of the protein. For each RBP, we performed two independent transductions of two different targeting shRNAs and two independent transductions of a non-targeting shRNA, or performed two independent transfections with a plasmid directing expression of the RBP as a V5-tagged fusion, with the FLAG construct as a control.

2020-08-18 | GSE117293 | GEO

Systematic identification of RNA-binding proteins and tethered domains that activate exon splicing inclusion [eCLIP-seq]

Project description:Systematic identification of RNA-binding proteins and tethered domains that activate exon splicing inclusion [eCLIP-seq]

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Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival [eCLIP-Seq]

Project description:Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using a recently developed enhanced UV crosslinking and immunoprecipitation (eCLIP) approach, we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region- and binding site-level IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3'UTR-enriched targets. RNA Bind-N-Seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and an increase in cell death. For cell adhesion, in hPSCs we find IMP1 maintains levels of integrin mRNA, specifically regulating RNA stability of ITGB5. Additionally, we show IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. eCLIP-seq was performed in biological replicate for IGF2BP1/IMP1 and IGF2BP2/IMP2, as well as one replicate each for IGF2BP3/IMP3, RBFOX2, and an IgG control. Each sample has a size-matched input control for analysis

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