Anti-RNF144B monoclonal antibody, clone 7C22

Ubiquitination is a post-translational modification of proteins that is widely present in eukaryotic cells. There is increasing evidence that ubiquitinated proteins play crucial roles in the immune response process. In mammals, RING-between-RING (RBR) proteins play a key role in regulating immune signaling as the important E3 ubiquitin ligases during ubiquitination. However, the function of RBR in fish is still unclear. In the present study, six RBR genes (RNF19A, RNF19B, RNF144AA, RNF144AB, RNF144B and RNF217) of grass carp (Ctenopharyngodon idellus) were cloned and characterized. Similar to mammals, all six members of RBR family contained RING, inbetween-ring (IBR) and transmembrane (TM) domains. These genes were constitutively expressed in all studied tissues, but the relative expression level differed. Following grass carp reovirus(GCRV) infection, the expression of six RBR genes in liver, gill, spleen and intestine significantly altered. Additionally, their expression in Ctenopharyngodon idellus kidney (CIK) cells was significantly increased after GCRV infection. And deficiency of RNF144B in CIK with small interference RNA (siRNA) up-regulated polyinosinic:polycytidylic acid poly(I:C))- induced inflammatory cytokines production, including 1FN-I, TNF-alpha, IL-6, and transcription factor IRF3, which demonstrated that RNF144B was a negative regulator of inflammatory cytokines. Our results suggested that the RBR might play a vital role in regulating immune signaling and laid the foundation for the further mechanism research of RBR in fishes.

Delta Np63 is a transcription factor that is critical for the development of stratified epithelia and is overexpressed or amplified in >80% of squamous cell carcinomas (SCCs). We identified the RING finger E3 ubiquitin ligase PIR2/Rnf144b as a direct transcriptional target of Delta Np63 alpha and showed that its expression parallels that of Delta Np63 alpha in keratinocytes, SCC cell lines and SCCs. We used primary keratinocytes as a model system to investigate the function of PIR2/Rnf144b in stratified epithelia. Depletion of PIR2/Rnf144b severely impaired keratinocyte proliferation and differentiation, associated with accumulation of p21(WAF1/CIP1); a known target of PIR2/Rnf144b. More importantly, we found that PIR2/Rnf144b binds and mediates proteasomal degradation of Delta Np63 alpha, generating a hitherto unknown auto-regulatory feedback loop. These findings substantiate PIR2/Rnf144b as a potentially critical component of epithelial homeostasis, acting downstream of Delta Np63 alpha to regulate cellular levels of p21(WAF1/CIP1) and Delta Np63 alpha.