A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days
A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days
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DOI：
10.1186/s12864-019-6106-2
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发表时间：
2019-10-21
期刊：
影响因子：
4.4
通讯作者：
Boiani, Michele
中科院分区：
文献类型：
--
作者：
Israel, Steffen;Casser, Ellen;Boiani, Michele
虽然DNA和RNA方法是破坏特定基因表达的常规方法，但完全了解发育过程还需要蛋白质方法，因为：卵母细胞和早期胚胎积累蛋白质，而这些不受DNA和RNA方法的直接影响。当卵母细胞中的蛋白质遇到特异性抗体和TRIM21泛素蛋白连接酶时，它们可以在蛋白酶体中降解，产生短暂的功能性敲除，从而揭示蛋白质的作用。然而，对于这种靶向蛋白水解能否成功地用于哺乳动物发育的研究，存在疑问，因为瞬时效应的持续时间尚不清楚，也因为递送的试剂量必须与靶蛋白的量足够，而靶蛋白的量也是未知的。结果我们发现，通过使用基于强度的绝对定量（iBAQ）算法的质谱法估计，小鼠卵细胞含有高达1E-02皮摩尔/蛋白质。然而，卵子只能容纳大约1E-04皮摩尔的抗体或TRIM21而不会产生毒性作用。在这个框架内，我们证明了trim21介导的蛋白质耗竭有效地破坏了滋养外胚层形成的胚胎过程，这一过程严重依赖于TEA结构域家族成员4 （Tead4）基因。在基因和蛋白表达恢复到基线之前，从1细胞阶段开始的TEAD4缺失持续3天。这段时间足够长，导致表型与已发表的零突变和RNA干扰研究完全一致：滋养外胚层基因Cdx2和Gata3明显表达不足，胚胎在子宫内空化和植入的能力严重受损。组学数据可通过ProteomeXchange （PXD012613）和GEO （GSE124844）获得。结论trim21介导的蛋白缺失是破坏小鼠发育过程中基因功能的有效手段，前提是靶基因的选择要谨慎，方法要准确。本研究中收集的知识为加速Tead4以外的其他基因的蛋白质消耗提供了基本的知识（先决条件、要求、限制）。
Background While 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. Results We 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 approximate to 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). Conclusions TRIM21-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.