Large-scale tethered function assays identify factors that regulate mRNA stability and translation
| Autor: | Sebastian Markmiller, Joshua L. Schwartz, David E. Hill, Gene W. Yeo, Archana Shankar, Jonathan C. Schmok, Gabriel A. Pratt, Duy B. Scaletta, Brian A. Yee, Stefan Aigner, Frederick E. Tan, Shashank Sathe, Jason L. Nathanson, Yuanchi Ha, En-Ching Luo |
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| Rok vydání: | 2020 |
| Předmět: |
Ultraviolet Rays
RNA Stability 1.1 Normal biological development and functioning Biophysics RNA-binding protein Biology Medical and Health Sciences Article Cell Line 03 medical and health sciences Open Reading Frames 0302 clinical medicine Structural Biology Underpinning research Protein biosynthesis Genetics Humans Luciferases Molecular Biology 3' Untranslated Regions 030304 developmental biology Regulation of gene expression 0303 health sciences Reporter gene Messenger RNA Binding Sites Three prime untranslated region RNA RNA-Binding Proteins Translation (biology) Biological Sciences Recombinant Proteins Cell biology Polyribosomes Protein Biosynthesis Chemical Sciences Generic health relevance CRISPR-Cas Systems Carrier Proteins 030217 neurology & neurosurgery Developmental Biology |
| Zdroj: | Nature structural & molecular biology, vol 27, iss 10 Nat Struct Mol Biol |
| Popis: | 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 3ʹ-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. A survey of human RNA-binding proteins based on luciferase-based 3ʹ-untranslated-region tethered function assays and identification of their target mRNAs provides insights into their role in RNA metabolism. |
| Databáze: | OpenAIRE |
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