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

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

PROVIDER: PRJNA482134 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7547730.fastq.gz	Fastqsanger.gz
	SRR7547731.fastq.gz	Fastqsanger.gz
	SRR7547732.fastq.gz	Fastqsanger.gz
	SRR7547733.fastq.gz	Fastqsanger.gz
	SRR7547734.fastq.gz	Fastqsanger.gz

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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 (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 (eCLIP)

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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.

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 (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.

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Project description:The half-life of mRNAs, as well as their translation, increases proportionally to the optimal codons, indicating a tight coupling of codon-dependent differential translation and degradation. Little is known about the regulation of this coupling. We found that the coupling in yeast displays a dichotomous behavior regarding the mRNA coding sequence length. Below a critical length, codon optimality fails to affect the stability of mRNAs even though they can be efficiently translated into short peptides and proteins. Above this threshold length, codon optimality-dependent differential mRNA stability emerges in a switch-like fashion, which coincides with a similar increase in the polysome propensity of the mRNAs. This threshold length can be tuned by the untranslated regions (UTR). Some of these UTRs can destabilize mRNAs without reducing translation, which plays a role in controlling the amplitude of the oscillatory expression of cell-cycle genes. Our findings help understand the translation of short peptides from noncoding RNAs and the translation by localized monosomes in neurons.

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Polysome propensity and tunable thresholds in coding sequence length enable differential mRNA stability

Project description:Polysome propensity and tunable thresholds in coding sequence length enable differential mRNA stability

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