CRISPR screening identifies TRIM27 as a destabilizer of the Smith–Magenis syndrome protein RAI1

Abstract The nervous system is highly sensitive to alterations in the dosage of genes crucial for neurodevelopment, as exemplified by retinoic acid-induced 1 (RAI1). A 50% change in RAI1 gene copy number, resulting in either reduced or increased protein levels, leads to distinct neurodevelopmental disorders. RAI1 haploinsufficiency causes Smith–Magenis syndrome (SMS), whereas RAI1 duplication underlies Potocki–Lupski syndrome (PTLS). We recently demonstrated that restoring Rai1 levels can improve SMS-related disease phenotypes in mice. However, despite protein stability being a major determinant of protein abundance, there are currently no therapeutic approaches to modulate RAI1 protein stability. Here, we performed a forward CRISPR screen in human cells to identify post-translational regulators of RAI1 steady-state levels and identified tripartite motif containing 27 (TRIM27) as a destabilizer of RAI1. We show that RAI1 degradation occurs primarily through the ubiquitin proteasome system, with TRIM27 interacting with RAI1 and enabling TRIM27-dependent lysine(K)48- and K63-linked RAI1 ubiquitination. Finally, in SMS mouse primary neurons, we demonstrate that knocking down TRIM27 partially rescues SMS-associated morphological phenotypes. Our findings provide the first mechanistic insight into RAI1 proteostasis and highlight TRIM27 as a potential therapeutic target for SMS, highlighting the potential of manipulating ubiquitin-mediated proteostasis to restore gene dosage altered by copy number variations.