The NEDD4-binding protein N4BP1 degrades mRNA substrates through the coding sequence independent of nonsense-mediated decay.

3'UTRs are recognized for their role in regulating mRNA turnover while the turnover of a specific group of mRNAs mediated by coding sequences (CDSs) remains poorly understood. N4BP1 is a critical inflammatory regulator in vivo with a molecular mechanism that is not yet clearly defined. Our study reveals that N4BP1 efficiently degrades its mRNA targets via CDS rather than the 3'-UTR. This CDS-dependent mRNA turnover mechanism appears to be a general feature of N4BP1, as evidenced by testing multiple mRNA substrates, such as Fos-C, Fos-B, Jun-B, and C-X-C motif chemokine ligand 1. Detailed mapping of the motif identified a crucial 33-nt (289-322) sequence near the 5'-end of Fos-C-CDS, where the presence of polyC is necessary for N4BP1-mediated degradation. Functional studies involving domain deletion and point mutations showed that both the K homology and N4BP1, YacP-like nuclease domains are essential for N4BP1 to restrict mRNA substrates. The function of N4BP1 in mRNA turnover is not dependent on nonsense-mediated decay as it efficiently restricts mRNA substrates even in cells deficient in UPF1, UPF3A, and UPF3B. Additionally, the function of N4BP1 is not reliant on LUC7L3 despite its known association with this protein. Our findings suggest that N4BP1 acts as an endoribonuclease to degrade mRNA substrates primarily through CDSs containing a C-rich motif.