Moon Hye Ji, Lee Na Yeon, Do Eun Kyoung, Lee Seo Yul, Park Gyu Tae, Lim Jae Kyong, Seo Jeong Kon, Kim Jae Ho
Gene and Cell Therapy Research Center for Vessel-Associated Diseases, Pusan National University, Yangsan, Republic of Korea.
Department of Physiology, School of Medicine, Pusan National University, Yangsan, Republic of Korea.
Stem Cells. 2022 Apr 29;40(4):385-396. doi: 10.1093/stmcls/sxac010.
Lin28A is an RNA-binding protein that controls mammalian development and maintenance of the pluripotency of embryonic stem cells (ESCs) via regulating the processing of the microRNA let-7. Lin28A is highly expressed in ESCs, and ectopic expression of this protein facilitates reprogramming of somatic cells to induced pluripotent stem cells. However, the mechanisms underlying the post-translational regulation of Lin28A protein stability in ESCs remain unclear. In the present study, we identified Kap1 (KRAB-associated protein 1) as a novel Lin28A-binding protein using affinity purification and mass spectrometry. Kap1 specifically interacted with the N-terminal region of Lin28A through its coiled-coil domain. Kap1 overexpression significantly attenuated Lin28A ubiquitination and increased its stability. However, small interfering RNA-mediated knockdown of Kap1 promoted the ubiquitination of Lin28A, leading to its proteasomal degradation. Trim71, an E3 ubiquitin ligase, induced Lin28A degradation and Kap1 knockdown accelerated the Trim71-dependent degradation of Lin28A. Mutation of the lysine 177 residue of Lin28A to arginine abrogated the ubiquitination and degradation of Lin28A which were accelerated by Kap1 silencing. Moreover, Kap1 overexpression led to the accumulation of Lin28A in the cytoplasm, but not in the nucleus, and reduced the levels of let-7 subtypes. These results suggest that Kap1 plays a key role in regulation of the stability of Lin28A by modulating the Trim71-mediated ubiquitination and subsequent degradation of Lin28A, thus playing a pivotal role in the regulation of ESC self-renewal and pluripotency.
Lin28A是一种RNA结合蛋白，它通过调控微小RNA let-7的加工过程来控制哺乳动物的发育以及胚胎干细胞（ESC）多能性的维持。Lin28A在ESC中高度表达，该蛋白的异位表达有助于将体细胞重编程为诱导多能干细胞。然而，ESC中Lin28A蛋白稳定性的翻译后调控机制仍不清楚。在本研究中，我们使用亲和纯化和质谱法鉴定出Kap1（KRAB相关蛋白1）是一种新型的Lin28A结合蛋白。Kap1通过其卷曲螺旋结构域与Lin28A的N端区域特异性相互作用。Kap1的过表达显著减弱了Lin28A的泛素化并增加了其稳定性。然而，小干扰RNA介导的Kap1敲低促进了Lin28A的泛素化，导致其通过蛋白酶体降解。E3泛素连接酶Trim71诱导Lin28A降解，而Kap1敲低加速了Trim71依赖的Lin28A降解。将Lin28A的赖氨酸177残基突变为精氨酸消除了由Kap1沉默加速的Lin28A的泛素化和降解。此外，Kap1过表达导致Lin28A在细胞质中积累，而非在细胞核中积累，并降低了let-7亚型的水平。这些结果表明，Kap1通过调节Trim71介导的Lin28A泛素化及随后的降解，在调控Lin28A稳定性中起关键作用，从而在ESC自我更新和多能性调控中起关键作用。
