Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons
- University of Edinburgh, United Kingdom
- Hospital for Sick Children, Canada
- Michigan State University, United States
Abstract
Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and condensin II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.
Data availability
Proteomics data underlying Figure 6B have been submitted to the PRIDE database under accession PXD032374. All other primary data, including flow cytometry files, fluorescence imaging and uncropped western blot scans are available through the DRYAD digital repository at https://doi.org/10.5061/dryad.g1jwstqt9. Requests for the Rosa26Tir1 transgenic mouse line should be addressed to Bin Gu (guibin1@msu.edu), and requests for the Ncaph- and Ncaph2-AID:Clover lines should be addressed to Andrew Wood (Andrew.j.wood@ed.ac.uk).
-
Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degronsDryad Digital Repository, doi:10.5061/dryad.g1jwstqt9.
Article and author information
Author details
Jennifer Margaret Brisbane
Alex von Kriegsheim
Funding
Medical Research Council
- Lewis Macdonald
- Gillian C Taylor
- Jennifer Margaret Brisbane
- Ersi Christodoulou
- Lucy Scott
- Andrew J Wood
Wellcome Trust (102560/Z/13/Z)
- Lewis Macdonald
- Gillian C Taylor
- Jennifer Margaret Brisbane
- Ersi Christodoulou
- Lucy Scott
- Andrew J Wood
Canadian Institutes of Health Research (JR FDN-143334)
- Janet Rossant
- Bin Gu
Medical Research Council (MC_PC_21040)
- Andrew J Wood
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Guillaume Pavlovic, PHENOMIN, Institut Clinique de la Souris (ICS), CELPHEDIA, France
Ethics
Animal experimentation: All animal work was approved by a University of Edinburgh internal ethics committee and was performed in accordance with institutional guidelines under license by the UK Home Office. AID knock-in alleles were generated under project license PPL 60/4424. Rosa26Tir1 knockin mouse lines were generated under the Canadian Council on Animal Care Guidelines for Use of Animals in Research and Laboratory Animal Care under protocols approved by the Centre for Phenogenomics Animal Care Committee (20-0026H). Experiments involving double transgenic animals were conducted under the authority of UK project license PPL P16EFF7EE
Version history
- Preprint posted: January 13, 2022 (view preprint)
- Received: February 18, 2022
- Accepted: May 19, 2022
- Accepted Manuscript published: June 23, 2022 (version 1)
- Version of Record published: July 11, 2022 (version 2)
Copyright
© 2022, Macdonald et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 6,407
- views
-
- 965
- downloads
-
- 15
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons
eLife 11:e77987.
https://doi.org/10.7554/eLife.77987
Further reading
-
- Cell Biology
- Chromosomes and Gene Expression
Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.
-
- Chromosomes and Gene Expression
Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.
