Control of Meiotic and Mitoic Progression by the F-Box Protein NIPA In Vivo.

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein (FBP) that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry.

Using a conditional knockout strategy we inactivated the gene encoding the FBP NIPA to determined the consequences of NIPA deletion in vivo. The targeting construct was designed to flank exon 1 and 2 of the NIPA gene with loxP-sites. Deletion of this region was obtained by crossing the floxed mice to a cre-transgenic mouse strain expressing cre ubiquitously. NIPA deficiency did not affect the viability of NIPA −/− animals. Mating of heterozygotes yielded NIPA +/+, NIPA +/− and NIPA −/− offspring approximately at the expected Mendelian ratio. Although copulatory behavior was normal and vaginal plugs were produced, NIPA-deficient animals have a fertility defect. 100% of the tested NIPA −/− males and 60% of NIPA −/− females never produced progeny with young fertile wild-type mice. Interestingly, histological evaluation showed progressive testis atrophy in NIPA −/− males. Further analyses indicate a block in germ cell differentiation at the stage of meiotic prophase, no spermatides or spermatozoa were observed in NIPA-deficient animals. High levels of nuclear cyclin B1, a previously reported NIPA substrate, were present in NIPA-deficient germ cells. Furthermore, inactivation of NIPA leads to premature mitotic entry and subsequent mitotic catastrophe and TUNEL positive apoptosis in germ cells. Long-term studies of NIPA deficient mice may display a potential role of NIPA in tumor development.

Since we found the most striking phenotype in high proliferating germ cells our results strongly confirm the cell cycle regulatory function of NIPA.