Alternative exon skipping biases substrate preference of the deubiquitylase USP15 for mysterin/RNF213, the moyamoya disease susceptibility factor

Abstract

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.

ALKBH5 regulates anti–PD-1 therapy response by modulating lactate and suppressive immune cell accumulation in tumor microenvironment

“…Usp15 affects signaling by transforming growth factor-β, which attracts and activates Tregs. Notably, Met and Usp15 are expressed as isoforms that have markedly different functions ( 43 , 44 ), suggesting that gene-splicing changes may play a role in TME composition and eventually affecting the immunotherapy efficacy. Taken together, these data indicate that Alkbh5 regulates the density of m 6 A near spice sites in multiple mRNAs with functions potentially important during GVAX/anti–PD-1 therapy.…”

The deubiquitylase USP15 regulates topoisomerase II alpha to maintain genome integrity

“…For example, during the cell cycle, periodic phosphorylation activates USP16 and USP3714,15 but inactivates USP8 through recruitment of 14-3-3 proteins16. The regulated expression of DUBs may also control their cellular availability, and alternative splicing can generate DUB isoforms that are targeted to distinct subcellular compartments, as described for USP3317, or exhibit different substrate specificity, as recently suggested for USP1518.…”

The AAA+ ATPase/ubiquitin ligase mysterin stabilizes cytoplasmic lipid droplets

“…Anti-human mysterin mouse monoclonal antibodies (1D6 and 1C9) were generated as previously described (kindly shared by Seiji Takashima, Graduate School of Medicine, Osaka University, Osaka, Japan; Kotani et al, 2017). Other antibodies were purchased, namely, anti-human PLIN3 mouse monoclonal antibody (sc-390169; Santa Cruz), anti-human ATGL rabbit polyclonal antibodies (for immunostaining, sc-67355; Santa Cruz; for immunoblot, #2138S; Cell Signaling), anti-human AUP1 rabbit polyclonal antibody (HPA007674; Atlas Antibodies), anti-human tubulin rabbit monoclonal antibody (41117; Millipore), anti-rabbit GAPDH mouse monoclonal antibody (5G4; Hy Test), and anti-FLAG M2 mouse monoclonal antibody (F1804; Sigma).…”

“…Ubiquitin ligases covalently modify substrate proteins with the small protein ubiquitin, resulting in their proteolysis or functional regulation (Metzger et al, 2014). Previous studies proposed that mysterin exerts ubiquitylation activity toward a variety of substrate proteins, including itself (autoubiquitylation; Liu et al, 2011; Banh et al, 2016; Scholz et al, 2016; Kotani et al, 2017). To our knowledge, mysterin is the only known protein that exerts both AAA+ ATPase and ubiquitin ligase activities, while how it coordinates the unique combination of enzymatic activities and what role it plays in cells remain elusive.…”