Abstract
SummaryMethods 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.
Highlights
Interfering with protein expression is a powerful strategy to investigate the function of a protein
A widely applicable protein depletion method that acts exclusively at the protein level is currently lacking. Such a method would allow the acute depletion of endogenous proteins, and the study of protein function in non-dividing primary cells in which DNA-targeting must be done at the whole animal level and RNA-targeting cannot deplete stable proteins
The E2 enzymes Ube2W and Ube2N/2V2 have been implicated in TRIM21-mediated degradation (Fletcher et al, 2015)
Summary
Interfering with protein expression is a powerful strategy to investigate the function of a protein. RNA-targeting methods such as RNAi are widely used to knockdown expression of a protein by destroying the mRNA (Elbashir et al, 2001). In both approaches, protein depletion is indirect and dependent on the inherent turnover of the protein. A widely applicable protein depletion method that acts exclusively at the protein level is currently lacking Such a method would allow the acute depletion of endogenous proteins, and the study of protein function in non-dividing primary cells in which DNA-targeting must be done at the whole animal level and RNA-targeting cannot deplete stable proteins
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