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An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans

Kelly Hills-Muckey, Michael A. Q. Martinez, Natalia Stec, Shilpa Hebbar, Joanne Saldanha, View ORCID ProfileTaylor N. Medwig-Kinney, Frances E. Q. Moore, Mariia Ivanova, Ana Morao, View ORCID ProfileJordan D. Ward, Eric G. Moss, View ORCID ProfileSevinc Ercan, Anna Y. Zinovyeva, View ORCID ProfileDavid Q. Matus, Christopher M. Hammell
doi: https://doi.org/10.1101/2021.08.06.455414
Kelly Hills-Muckey
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
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Michael A. Q. Martinez
2Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA
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Natalia Stec
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
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Shilpa Hebbar
3Kansas State University, Division of Biology, Manhattan, KS 66506, USA
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Joanne Saldanha
2Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA
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Taylor N. Medwig-Kinney
2Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA
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  • ORCID record for Taylor N. Medwig-Kinney
Frances E. Q. Moore
2Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA
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Mariia Ivanova
4Department of Molecular Biology, Rowan University, Stratford, NJ 08084, USA
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Ana Morao
5New York University, Center for Genomics and Systems Biology, 100 Washington Square East, 852 Brown, New York, NY, 10003, USA
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Jordan D. Ward
6Department of Molecular, Cell, and Developmental Biology, University of California-Santa Cruz, Santa Cruz, CA 95064, USA
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Eric G. Moss
4Department of Molecular Biology, Rowan University, Stratford, NJ 08084, USA
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Sevinc Ercan
5New York University, Center for Genomics and Systems Biology, 100 Washington Square East, 852 Brown, New York, NY, 10003, USA
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Anna Y. Zinovyeva
3Kansas State University, Division of Biology, Manhattan, KS 66506, USA
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David Q. Matus
2Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA
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Christopher M. Hammell
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
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  • For correspondence: chammell@cshl.edu
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ABSTRACT

The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex (SKR-1/2-CUL-1-F-box (SCF)), targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin-derivative/mutant AtTIR1 pair (C. elegans AID version 2 (C.e.AIDv2)) that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID targets, addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expands the utility of the AID system and broadens the number of proteins that can be effectively targeted with it.

ARITCLE SUMMARY Implementation of the auxin induced degradation (AID) system has increased the power if the C. elegans model through its ability to rapidly degrade target proteins in the presence of the plant hormone auxin (IAA). The current C.e.AID system is limited in that a substantial level of target degradation occurs in the absence of ligand and full levels of target protein degradation require high levels of auxin inducer. In this manuscript, we modify the AID system to solve these problems.

Competing Interest Statement

The authors have declared no competing interest.

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Posted August 09, 2021.
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An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans
Kelly Hills-Muckey, Michael A. Q. Martinez, Natalia Stec, Shilpa Hebbar, Joanne Saldanha, Taylor N. Medwig-Kinney, Frances E. Q. Moore, Mariia Ivanova, Ana Morao, Jordan D. Ward, Eric G. Moss, Sevinc Ercan, Anna Y. Zinovyeva, David Q. Matus, Christopher M. Hammell
bioRxiv 2021.08.06.455414; doi: https://doi.org/10.1101/2021.08.06.455414
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An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans
Kelly Hills-Muckey, Michael A. Q. Martinez, Natalia Stec, Shilpa Hebbar, Joanne Saldanha, Taylor N. Medwig-Kinney, Frances E. Q. Moore, Mariia Ivanova, Ana Morao, Jordan D. Ward, Eric G. Moss, Sevinc Ercan, Anna Y. Zinovyeva, David Q. Matus, Christopher M. Hammell
bioRxiv 2021.08.06.455414; doi: https://doi.org/10.1101/2021.08.06.455414

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