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

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 (SDE2UBL) releases the biologically functional C-terminal domain (SDE2CT), 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 SDE2UBL 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 SDE2UBL 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.