Abstract
Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3′ UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism.
Highlights
Techniques for precise temporal and spatial control of protein expression enable detailed analysis of developmental mechanisms
Design strategy for the auxin-inducible degradation (AID) system in C. elegans TIR1-dependent protein degradation has been most extensively characterized in the model plant Arabidopsis thaliana
We constructed a synthetic TIR1 gene that was codon optimized for C. elegans and contains two introns (Fig. S1B)
Summary
Techniques for precise temporal and spatial control of protein expression enable detailed analysis of developmental mechanisms. Depletion of gene products in specific stages and tissues has been achieved through RNAi (Qadota et al, 2007), or by gene disruption via tissue-specific expression of sequence-specific nucleases (Cheng et al, 2013; Shen et al, 2014). These approaches are indirect and irreversible, as they rely on inactivation of a gene or on mRNA degradation. There is often a substantial lag between induction and protein depletion, the duration of which depends on mRNA and/or protein stability (Elbashir et al, 2001; Fire et al, 1998)
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