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

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Homo sapiens

PROVIDER: GSE117294 | GEO | 2020/08/18

REPOSITORIES: GEO

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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.

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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 (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

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

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 (eCLIP)

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

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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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