Montreal Clinical Research Institute (IRCM), Montreal, Quebec, Canada; Molecular Biology Programs, Université de Montréal, Montreal, Quebec, Canada; Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
Montreal Clinical Research Institute (IRCM), Montreal, Quebec, Canada.
J Biol Chem. 2023 Sep;299(9):105123. doi: 10.1016/j.jbc.2023.105123. Epub 2023 Aug 1.
Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionarily conserved and well-established roles of MOB1 (MOB1A/B) in regulating tissue homeostasis within the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions and help assemble complexes is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification approach to define the interactomes of all seven human MOB proteins in HeLa and human embryonic kidney 293 cell lines. We uncovered >200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique association between MOB3C and 7 of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5' maturation. As a proof of principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.
除了 MOB1（MOB1A/B）在 Hippo 通路中调节组织内稳态的进化保守和成熟作用之外，单极纺锤体结合蛋白（MOB）家族成员介导的不同功能仍然难以捉摸。由于 MOB 蛋白是衔接蛋白，了解它们如何参与蛋白-蛋白相互作用并帮助组装复合物对于定义它们的全部生物学功能至关重要。为了解决这个问题，我们采用了一种邻近依赖性生物素鉴定方法，在 HeLa 和人胚肾 293 细胞系中鉴定了所有 7 种人类 MOB 蛋白的相互作用组。我们发现了 >200 种相互作用，其中至少 70%在 BioGrid 上没有报道。生成的数据集可靠地召回了研究充分的 MOB 的真正相互作用因子。我们进一步在亚家族和个体水平上定义了不同 MOB 之间的共同和差异相互作用组。我们发现 MOB3C 与 RNase P 复合物的 10 个蛋白亚基中的 7 个之间存在独特的关联，RNase P 复合物是一种催化 tRNA 5'成熟的内切酶。作为生成数据集稳健性的原理证明，我们通过使用亲和纯化-质谱法和 MOB3C 下拉物的 pre-tRNA 切割测定验证了 MOB3C 与催化活性的 RNase P 的特异性相互作用。总之，我们的数据为 MOB 蛋白的生物学提供了新的见解，并揭示了 MOB3C 的第一个相互作用因子，即 RNase P 复合物的组成部分，因此与 RNA 生物学有一个令人兴奋的交点。
