The mIAA7 degron improves auxin-mediated degradation in C. elegans

Abstract

Auxin-inducible degradation (AID) is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the AID system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in C. elegans, as previously reported in human cells. We tested depletion of a series of proteins with various sub-cellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for five out of six proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID* degron sequence. The mIAA7 degron also increased the leaky degradation for two of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system (C.e.AID2), which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the AID system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.

An expandable FLP-ON::TIR1 system for precise spatiotemporal protein degradation inC. elegans

“…Although mIAA7-dependent depletion of FOS-1A did not completely recapitulate the null allele phenotype, it significantly improved both the degradation kinetics and associated invasion defects following loss of a transcription factor with single cell specificity. This is in support of the first report using the mIAA7 degron in C. elegans, which emphasized that nuclear localized targets are more resistant to auxin-induced degradation (Sepers et al 2022). To further explore how protein abundance and subcellular localization of a target protein may affect degradation kinetics at singlecell resolution in a functional assay, we generated two new strains to compare the mIAA7 and AID degron kinetics following depletion of arx-2, the sole Arp2 subunit of the actin-related protein-2/3 (Arp2/3) complex in C. elegans (Sawa et al 2003).…”

“…The copyright holder for this preprint this version posted October 16, 2022. ; https://doi.org/10.1101/2022.10.14.512315 doi: bioRxiv preprint A recent report in C. elegans demonstrated that an alternative degron sequence, the mIAA7 degron, displayed faster degradation kinetics when paired with At TIR1 expression and auxin treatment (Sepers et al 2022). Compared to the minimal AID epitope, the mIAA7 sequence contains the 44aa domain required for At TIR1 recognition and additional N-terminal flanking sequences from the IAA protein that improve the interactions with TIR1 (Gray et al 2001;Ramos et al 2001) (Fig.…”

A reevaluation of the relationship between EGL-43 (EVI1/MECOM) and LIN-12 (Notch) duringC. elegansanchor cell invasion

“…The auxin-inducible degron (AID) system enables rapid degradation of C. elegans proteins (Ashley et al, 2021; Hills-Muckey et al, 2021; Martinez et al, 2020; Negishi et al, 2021; Sepers et al, 2022; Zhang et al, 2015). It requires a minimal AID tag on the protein of interest (POI), expression of the Arabidopsis F-box protein TIR1, and exogenous exposure to the plant hormone auxin.…”

Cell cycle perturbation uncouples mitotic progression and invasive behavior in a post-mitotic cell

“…2B,F). These ambiguities could be addressed in future studies by creating and/or combining conditional null alleles using the auxin-inducible degron (AID) system (Ashley et al, 2021;Hills-Muckey et al, 2021;Martinez et al, 2020;Negishi et al, 2021;Sepers et al, 2022;Xiao et al, 2023; or an analogous approach (Nance and Frøkjaer-Jensen, 2019).…”