USP5 acts as a proteolytic switch to control SDE2 function via UBL-directed cleavage

Abstract Proteolytic processing is a fundamental regulatory mechanism in eukaryotic cells, yet the molecular identities and mechanisms underlying such events are often poorly defined. Silencing Defective 2 (SDE2), an essential human protein, plays important roles in mRNA splicing, DNA repair and ribosomal biogenesis. Cleavage of SDE2 downstream to its N-terminal ubiquitin-like domain (SDE2 UBL ) releases the biologically functional C-terminal domain (SDE2 CT ), highlighting the importance of this proteolytic event. However, the protease responsible for this cleavage in human cells has remained undefined. Here, we identify deubiquitinating enzyme, ubiquitin-specific protease 5 (USP5), as the selectively primary effector of SDE2 cleavage both in vitro and in cell. Biophysical and structural analysis suggests that SDE2 UBL engages with USP5 through a two-site interaction that mirrors key features of ubiquitin recognition, supporting a mechanism of substrate mimicry. Functionally, depletion of USP5 increases intron retention in previously reported SDE2-dependent transcripts, linking the removal of SDE2 UBL domain by USP5 to the role of SDE2 in mRNA splicing. Together, these findings reveal a non-canonical proteolytic function of USP5, uncovering a previously unrecognised regulatory axis linking deubiquitinating enzymes to protein maturation, expanding the substrate repertoire for USP5 and providing a framework for identifying protease–substrate relationships in post-translational regulation.