Presentation/Session Information

Session Information

Session Title: RNA Interference, Noncoding RNAs, and Genetic Technologies Session Type: Parallel
Session Location: Carnesale Palisades Ballroom Session Time: Sat, Jun 27 8:30AM - 11:30AM

Presentation Information

Program Number: 149 Presentation Time: 11:06AM - 11:18AM

Presentation Content

An auxin-inducible degradation (AID) system for precise temporal and spatial control of protein depletion.L. Zhang 1,2,3,4, J. Ward 5, Z. Cheng 1,2,3,4, A. Dernburg 1,2,3,4. 1)Department of Molecular and Cell Biology, Howard Hughes Medical Institute, Berkeley, CA 94720-3220, USA; 2)Howard Hughes Medical Institute; 3)Life Sciences Division, Department of Genome Dynamics, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; 4)California Institute for Quantitative Biosciences; 5)Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA

Established approaches for protein depletion in C. elegans have primarily relied on disrupting genes or inhibiting their expression. A method for posttranslational depletion of proteins has recently been developed (Armenti et al., 2014), but this approach has some limitations: In particular, it cannot be used in the germline or in early embryos, and conditional depletion involves induction of a ubiquitin ligase, requiring both heat shock and lag time. As an alternative method for rapid, inducible protein depletion, we have implemented the auxin-inducible degradation (AID) system in C. elegans. This system relies on expression of a plant-specific F-box protein, TIR1, which interacts with endogenous Skp1 and Cul1 proteins. This SCF ubiquitin ligase complex recognizes substrates only in the presence of auxin (indole acetic acid, or IAA). We expressed an optimized TIR1 transgene and found that it mediates rapid, auxin-dependent degradation of nuclear or cytoplasmic proteins tagged with a short (44-amino acid) degron sequence. The degron can be fused to endogenous genes or transgenes, with or without a fluorescent protein or other tag as a reporter for expression and degradation. CRISPR/Cas9 editing with pha-1(ts) co-conversion (Ward, 2015) allowed efficient introduction of degron-3xFLAG tags in 8-9 days. TIR1 expression or IAA alone had no detectable effects on worm viability or fertility. The AID system mediates inducible protein degradation in somatic tissues, embryos, and the mitotic and/or meiotic germline. Degron-tagged proteins are depleted within 30 minutes in the soma or an hour in the germline. Using MosSCI, we have engineered TIR1 transgenes with various promoter and 3’UTR sequences to restrict expression to specific somatic tissues or subregions within the germline. We have harnessed the system both to explore the role of dynamically expressed nuclear hormone receptors in molting and to analyze meiosis-specific roles for proteins required for germline proliferation. Thus, the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in C. elegans.

References: Armenti, S.T., Lohmer, L.L., Sherwood, D.R., and Nance, J. (2014). Development 141: 4640-7.




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