Expression pattern and functional analysis of the two RING box protein RBX in spermatogenesis of Chinese mitten crab Eriocheir sinensis.

Studies in E. sinensis have shown that ubiquitination mediated by Cullin-RING E3 ligases (CRLs) plays important roles in spermatogenesis. In other species, CRLs are also essential in cell cycle progression, DNA replication, signal transduction, gene transcription, and development. The catalytic RING component, the RING box protein, is an important part of CRLs. However, there have been few studies on CRLs in crustaceans. In this study, we cloned two RING box protein genes from the Chinese mitten crab, Eriocheir sinensis, termed Es-RBX1 and Es-RBX2 The full length Es-RBX1 cDNA comprises 741 nucleotides, and encodes a protein of 124 amino acid residues, whereas the Es-RBX2 cDNA comprises 1325 nucleotides, and encodes a protein of 110 amino acid residues. Bioinformatics analysis showed that the domains and structure of the RBX proteins have been highly conserved during evolution. Quantitative real-time polymerase chain reaction and western blotting showed that Es-RBX1 is highly expressed in the testis, particularly during the spermatocyte stage, whereas Es-RBX2 did not show specific expression in the male reproductive system. Furthermore, Es-RBX1 is mainly distributed in the nucleus, and changed its location with the development of the nucleus. Co-immunoprecipitation showed that Es-RBX1 could bind Cullin4. These results suggested that Es-RBX1 plays a key role in spermatogenesis of E. sinensis though forming a complex with Cullin4.