Methods for Rapid Protein Depletion in C. elegans using Auxin-Inducible Degradation.
Methods for Rapid Protein Depletion in C. elegans using Auxin-Inducible Degradation.
复制标题
快速去除C. elegans使用生长素诱导降解。
DOI：
10.1002/cpz1.16
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发表时间：
2021-03
期刊：
影响因子：
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通讯作者：
Wignall SM
中科院分区：
文献类型：
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作者：
Divekar NS;Horton HE;Wignall SM
已经在模型系统中开发了许多方法来消耗或去除蛋白质，以阐明其功能作用。In C.在线虫中，用于蛋白质去除的常用方法是RNA干扰（RNAi），其中mRNA被靶向降解。此外，C.线虫是一种强大的遗传生物，适合大规模遗传筛选和CRISPR介导的基因组编辑。然而，这些方法在很大程度上导致组成性抑制，这可能使研究发育所必需的蛋白质或剖析动态细胞过程变得困难。因此，最近一直在努力开发快速去除或耗尽蛋白质的方法，以克服这些障碍。其中一种被证明是非常强大的方法是生长素诱导降解（AID）。为了在C.在线虫中，将44个氨基酸的降解决定子标签添加到目的蛋白质中，并在靶组织中表达拟南芥泛素连接酶TIR 1。然后，加入植物激素生长素;生长素介导TIR 1和degron标记的感兴趣的蛋白质之间的相互作用，这引发了其通过蛋白酶体的快速降解。在这里，我们概述了多种方法来诱导生长素介导的目标蛋白在C。elegans，突出了这种方法的多功能性和力量。
Numerous methods have been developed in model systems to deplete or inactivate proteins, to elucidate their functional roles. In C. elegans, a common method for protein depletion is RNA interference (RNAi), in which mRNA is targeted for degradation. Additionally, C. elegans is a powerful genetic organism, amenable to large scale genetic screens and CRISPR-mediated genome editing. However, these approaches largely lead to constitutive inhibition, which can make it difficult to study proteins essential for development or to dissect dynamic cellular processes. Thus, there have been recent efforts to develop methods to rapidly inactivate or deplete proteins, to overcome these barriers. One such method that is proving to be exceptionally powerful is auxin-inducible degradation (AID). In order to administer AID in C. elegans, a 44 amino acid degron tag is added to the protein of interest and an Arabidopsis ubiquitin ligase, TIR1, is expressed in target tissues. Then, the plant hormone auxin is added; auxin mediates an interaction between TIR1 and the degron-tagged protein of interest, which triggers its rapid degradation via the proteasome. Here, we have outlined multiple methods to induce auxin-mediated depletion of target proteins in C. elegans, highlighting the versatility and power of this method.