Kap1 Regulates the Stability of Lin28A in Embryonic Stem Cells

Abstract

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.