Analysis Of The Interactome And The N-terminal Function Of JMJD5

As a member of JmjC family, JMJD5(also named KDM8) has recently been reported to play a critical role in the regulation of cell cycle, embryonic development, osteoclastogenesis and circadian systems. Although JMJD5has been identified as a demethylase and also a hydroxylase, its specific substrates and regulatory mechanisms are still not clear. Meanwhile, the JmjC domain of JMJD5is located on the proximal C-terminus (amino acids270-416), and only occupies one-third of the protein sequence, while the N-terminal structure and function of JMJD5still remain largely unknown. To further study the biological function of JMJD5, we performed co-immunoprecipitation (Co-IP) combined with mass spectrometry to study its interactome, and38proteins were successfully identified. Their functions include cell signal transduction, RNA processing, DNA replication, cell cycle regulation, transcriptional regulation, apoptosis, etc. Our results indicate that JMJD5may has more complicated and important intracellular functions than we thought. Among those identified proteins, two nuclear transporters importin (31and transportin-1were found. Further studies showed that the N-terminus of JMJD5contains a classical nuclear localization signal (NLS)(amino acids152-170), which can be recognized by importin α/β nuclear transport system. Besides, transportin-1, a member of importin P2family, can bind to amino acids134-152in the N-terminal of JMJD5, also contributed to the nuclear transport of this protein. We further demonstrated that the NLS of JMJD5is necessary for cyclin A1transcription. In addition, we also found an nuclear export signal (NES)(amino acids34-38) in the N-terminal of JMJD5, recognized by the transporter protein CRM-1for mediating its nuclear export. Furthermore, we also found that JMJD5could directly interact with the DBD domain of tumor suppressor protein p53, thus inhibited p53transcriptional activity and the expression of target genes such as CDKN1A. The interaction of JMJD5and p53reduced after DNA damage, accompanied with the decreased chromatin binding JMJD5. These results indicate that JMJD5may play an important role in the DNA damage responses. In this study, we identified the interactome of JMJD5and studied the structure and function of its N-terminal domains. Our results provide a comprehensive understanding of JMJD5intracellular functions and its regulatory mechanisms.