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A method for the acute and rapid degradation of endogenous protein

ABSTRACT: A method for the acute and rapid degradation of endogenous protein

PROVIDER: PRJNA415216 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6197710.fastq.gz	Fastqsanger.gz
	SRR6197720.fastq.gz	Fastqsanger.gz
	SRR6197711.fastq.gz	Fastqsanger.gz
	SRR6197721.fastq.gz	Fastqsanger.gz
	SRR6197712.fastq.gz	Fastqsanger.gz

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A method for the acute and rapid degradation of endogenous protein

Project description:We developed Trim-Away as a a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away is able to remove unmodified native proteins within minutes of application by making use of the TRIM21 ubiquitin ligase that regognises antibody-protein complexes and harnesses the cellular degradation machinery. We performed transcriptome profiling (RNAseq) on NIH3T3 and NIH3T3-mCherry-mTRIM21 cells following electroporation with non-targeting antibody (IgG), bovine serum albumin (BSA), or buffer (PBS). We found that a total of only 8 protein-encoding transcripts were consistently downregulated more than 2-fold in the NIH3T3-mCherry-mTRIM21 cells (Kdm5d, Ddx3y, Eif2s3y, Uty, Asb4, Fat4, Papss2 and Lama2), although we cannot rule out the possibility that this is an indirect consequence of lentivirus construct integration rather than a direct consequence of TRIM21 overexpression. Notably, transcripts encoding previously described ligands of TRIM21, IRF-3, IRF-5, Skp2, DAXX, DDX41, and SQSTM1 were not significantly increased or decreased upon overexpression of TRIM21. Our experiments suggest that the cells overexpressing TRIM21 behave similar to wild-types.

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Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

Project description:Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. Using mouse genetics, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase subunit Tir1. Rapid degradation of condensin I and condensin II – two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

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A Method for the Acute and Rapid Degradation of Endogenous Proteins.

Project description:Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.

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