Mysterin, also known as RNF213, is an intracellular protein that forms large toroidal oligomers. Mysterin was originally identified in genetic studies of moyamoya disease (MMD), a rare cerebrovascular disorder of unknown etiology. While mysterin is known to exert ubiquitin ligase and putative mechanical ATPase activities with a RING finger domain and two adjacent AAA+ modules, its biological role is poorly understood. Here, we report that mysterin is targeted to lipid droplets (LDs), ubiquitous organelles specialized for neutral lipid storage, and markedly increases their abundance in cells. This effect was exerted primarily through specific elimination of adipose triglyceride lipase (ATGL) from LDs. The ubiquitin ligase and ATPase activities of mysterin were both important for its proper LD targeting. Notably, MMD-related mutations in the ubiquitin ligase domain of mysterin significantly impaired its fat-stabilizing activity. Our findings identify a unique new regulator of cytoplasmic LDs and suggest a potential link between the pathogenesis of MMD and fat metabolism.

The deubiquitylating enzyme USP15 plays significant roles in multiple cellular pathways including TGF-β signaling, RNA splicing, and innate immunity. Evolutionarily conserved skipping of exon 7 occurs during transcription of the mRNAs encoding USP15 and its paralogue USP4, yielding two major isoforms for each gene. Exon 7 of USP15 encodes a serine-rich stretch of 29 amino acid residues located in the inter-region linker that connects the N-terminal putative regulatory region and the C-terminal enzymatic region. Previous findings suggested that the variation in the linker region leads to functional differences between the isoforms of the two deubiquitylating enzymes, but to date no direct evidence regarding such functional divergence has been published. We found that the long isoform of USP15 predominantly recognizes and deubiquitylates mysterin, a large ubiquitin ligase associated with the onset of moyamoya disease. This observation represents the first experimental evidence that the conserved exon skipping alters the substrate specificity of this class of deubiquitylating enzymes. In addition, we found that the interactomes of the short and long isoforms of USP15 only partially overlapped. Thus, USP15, a key gene in multiple cellular processes, generates two functionally different isoforms via evolutionarily conserved exon skipping.