A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days

Steffen Israel,Georg Fuellen,Ellen Casser,Hannes C.A Drexler,Michele Boiani

doi:10.1186/s12864-019-6106-2

Steffen Israel, Georg Fuellen + Show 3 more

Open Access

https://doi.org/10.1186/s12864-019-6106-2

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Abstract

BackgroundWhile DNA and RNA methods are routine to disrupt the expression of specific genes, complete understanding of developmental processes requires also protein methods, because: oocytes and early embryos accumulate proteins and these are not directly affected by DNA and RNA methods. When proteins in the oocyte encounter a specific antibody and the TRIpartite Motiv-containing 21 (TRIM21) ubiquitin-protein ligase, they can be committed to degradation in the proteasome, producing a transient functional knock-out that reveals the role of the protein. However, there are doubts about whether this targeted proteolysis could be successfully used to study mammalian development, because duration of the transient effect is unknown, and also because amounts of reagents delivered must be adequate in relation to the amount of target protein, which is unknown, too.ResultsWe show that the mouse egg contains up to 1E-02 picomoles/protein, as estimated by mass spectrometry using the intensity-based absolute quantification (iBAQ) algorithm. However, the egg can only accommodate ≈1E-04 picomoles of antibody or TRIM21 without incurring toxic effects. Within this framework, we demonstrate that TRIM21-mediated protein depletion efficiently disrupts the embryonic process of trophectoderm formation, which critically depends on the TEA domain family member 4 (Tead4) gene. TEAD4 depletion starting at the 1-cell stage lasts for 3 days prior to a return of gene and protein expression to baseline. This time period is long enough to result in a phenotype entirely consistent with that of the published null mutation and RNA interference studies: significant underexpression of trophectodermal genes Cdx2 and Gata3 and strongly impaired ability of embryos to cavitate and implant in the uterus. Omics data are available via ProteomeXchange (PXD012613) and GEO (GSE124844).ConclusionsTRIM21-mediated protein depletion can be an effective means to disrupt gene function in mouse development, provided the target gene is chosen carefully and the method is tuned accurately. The knowledge gathered in this study provides the basic know-how (prerequisites, requirements, limitations) to expedite the protein depletion of other genes besides Tead4.

Highlights

While DNA and RNA methods are routine to disrupt the expression of specific genes, complete understanding of developmental processes requires protein methods, because: oocytes and early embryos accumulate proteins and these are not directly affected by DNA and RNA methods
Operating criterion no.1 for successful TRIpartite Motiv-containing 21 (TRIM21)-mediated protein depletion: known amount of target protein In order to be feasible in mammalian development for the study of gene functions, TRIM21-mediated protein depletion needs to be adapted to a cell type that is specialized to store proteins in large amounts and release them over days: the fertilized oocyte, or zygote
The intensity-based absolute quantification algorithm divides the sum of all precursor-peptide intensities by the number of theoretically observable peptides for the corresponding protein [35, 36]. iBAQ values are approximately proportional to the number of moles of protein present and iBAQi/ΣjiBAQj is the relative molar amount of protein ‘i’ among all proteins ‘j’, called relative iBAQ, briefly riBAQ [37]

Summary

Introduction

While DNA and RNA methods are routine to disrupt the expression of specific genes, complete understanding of developmental processes requires protein methods, because: oocytes and early embryos accumulate proteins and these are not directly affected by DNA and RNA methods. E.g. Pou5f1 (Oct4), null embryos were able to form blastocysts only to die shortly after implantation [3] These phenotypes were reproduced by inhibiting the mRNA via RNA interference or morpholino, as shown for Tead itself [4, 5] and its target gene Cdx2 [6]. Apart from exceptional cases of proteins with half-lives ranging from months to years [7], some embryonic proteins remain there for days after the cognate mRNA has been degraded (e.g. NLRP2 and members of the subcortical maternal complex, SCMC [8, 9]) These considerations fuel speculation that some null mutant phenotypes might be only partly revealed by DNA and RNA methods. Depletion has been conducted in Zebrafish via microinjection in the egg yolk, producing phenotypes in the embryos [22]

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