Rice (Oryza sativa) TIR1 and 5′adamantyl-IAA Significantly Improve the Auxin-Inducible Degron System in Schizosaccharomyces pombe

Abstract

The auxin-inducible degron (AID) system is a powerful tool to induce targeted degradation of proteins in eukaryotic model organisms. The efficiency of the existing Schizosaccharomyces pombe AID system is limited due to the fusion of the F-box protein TIR1 protein to the SCF component, Skp1 (Skp1-TIR1). Here, we report an improved AID system for S. pombe that uses the TIR1 from Oryza sativa (OsTIR1) not fused to Skp1. Furthermore, we demonstrate that degradation efficiency can be improved by pairing an OsTIR1 auxin-binding site mutant, OsTIR1F74A, with an auxin analogue, 5′adamantyl-IAA (AID2). We provide evidence for the enhanced functionality of the OsTIR1 AID and AID2 systems by application to the essential DNA replication factor Mcm4 and to a non-essential recombination protein, Rad52. Unlike AID, no detectable auxin-independent depletion of AID-tagged proteins was observed using AID2.

An improved auxin-inducible degron system for fission yeast

“…We showed in this study that, when paired with OsTIR1-F74A, 5-adamantyl-IAA is a highly effective inducer for the AID system and can minimize side effects on the transcriptome. Several recently published studies have also demonstrated that a bulky auxin analog, when paired with a mutant form of OsTIR1 with an enlarged auxin-binding pocket, acts as an effective AID inducer at much lower concentrations than auxin ( Nishimura et al 2020 ; Yesbolatova et al 2020 ; Watson et al 2021 ). When we initiated this study in 2018, 5-adamantyl-IAA was not commercially available and therefore we obtained it by chemical synthesis.…”

“…During the preparation of our manuscript, another study on improving the AID system for S. pombe was published ( Watson et al 2021 ). Similar to our findings, Watson et al also showed that OsTIR1 not fused with Skp1 can be expressed from a strong promoter without causing toxicity and is more effective in inactivating mcm4 than the Padh15-skp1-AtTIR1-NLS cassette of the Kanke system.…”

“…Understanding gene functions is a core pursuit of biological research. Our improved AID system is a new addition to a long list of tools and methods for creating conditional loss-of-function mutants in S. pombe ( Basi et al 1993 ; Rajagopalan et al 2004 ; Boe et al 2008 ; Kearsey and Gregan 2009 ; Kanke et al 2011 ; Tang et al 2011 ; Pai et al 2012 ; Watson et al 2013 ; Ding et al 2014 ; Watson et al 2021 ). This system will facilitate the analysis of gene functions in S. pombe , especially the functions of essential genes.…”

Applying the auxin-based degron system for the inducible, reversible and complete protein degradation in Komagataella phaffii

“…Auxin-induced degradation of Wsc1 in a wsc2 Δ wsc3 Δ mutant strain resulted in severe growth defects when applying OsTIR1 and AID2, whereas Wsc1 was degraded rapidly and efficiently under all conditions tested ( Figure 3 ). The biggest advantage of AID2 clearly is the reduced basal degradation of the target protein ( Watson et al., 2021 ; Yesbolatova et al., 2020 ). Although basal degradation of Wsc1 was fully absent when applying AID2 ( Figure 3 D), the growth phenotype observed for auxin-induced degradation of Wsc1 in a wsc2-3 Δ double delete strain was similar for AID (using 1-NAA and IAA) and AID2 (using 5-Ph-IAA).…”

Fission yeast Srr1 and Skb1 promote isochromosome formation at the centromere

“…4b), suggesting synthetic growth defects of the srr1∆ rad52∆ double deletion. To confirm this, we depleted Rad52 in srr1∆ cells using the auxininduced degron 2 (AID2) system 43 . In the presence of the F-box protein OsTIR1 F74A , an auxin analog 5'-adamantyl-IAA (5'a-IAA) induces polyubiquitin-dependent degradation of the AIDtagged Rad52 protein, Rad52-AID.…”