• RNF126 is identified as the E3 ubiquitin ligase for MIDN, catalyzing its ubiquitination at non-canonical cysteine, serine, and threonine residues.
• Non-lysine ubiquitination of MIDN targets it for 26S proteasomal degradation, revealing a novel regulatory mechanism.
• The RNF126-MIDN axis controls EGR1 abundance, thereby modulating tumor-suppressor proteins PTEN and p53.
• The RNF126-MIDN ubiquitination cascade represents a potential therapeutic target in testicular germ-cell tumors (TGCTs).