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

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. 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.