Molecular Characterization And Function Study Of BRCA1 Associated Protein 1(BAP1) In Artemia

Artemia, also named brine shrimp, possess powerful adaptations to stresses. It has evolved two reproductive pathways to cope with different environments, the ovoviviparous pathway, which gives birth to swimming nauplii, and the oviparous pathway, which releases silent encysted embryos. As it is known, the encysted embryos are developmentally arrested at the gastrula stage with a low metabolic rate and cells inside the embryo arrested at G1 phase as to our observation. When the silence is broken, drastic events would occur inside the embryo, such as, deposited energy mobilization, RNA and protein synthesis restoration, cellular differentiation, and morphological changing. However, during the development of the encysted embryos, no cell division was observed before emergence and the number of nuclei remains at about 4000 per embryo. The mechanism of the formation of the encysted embryo and its unusual developmental pattern is still elusive.The present research focused on the BRCA related protein 1(BAP1) in Artemia, investigating its possible roles in the formation and developmental process of the encysted embryos. BAP1, known as one ubiquitin carboxyl terminal hydrolase, belongs to the deubiquitinating enzyme family. It is reported that BAP1 participate in the regulation of cell cycle via its interaction with some transcript factors.In this research, the molecular structure of BAP1 in Artemia was characterized and was demonstrated to be belonging to the UCHs. Then the spatial-temporal expression of BAP1 was also characterized. It was showed that, in the long-light treated Artemia, BAP1 levels were nearly identical during the developmental stages. While in the short-light treated samples, BAP1 was especially abundant in the abodominal parts of Aretemia in mature stages. Then, Virtual Northern was carried out to identify BAP1 levels during the development of encyst embryos. It was found that BAP1 levels increased sharply just before the beginning of the mitogenesis.To investigate the roles of BAP1 in Aretemia, dsRNA interference was conducted. No apparent difference was observed between the encyst embryos produced by BAP1 RNAi group and control group under dissection microscope. By Western blotting analysis, it was found that the level of cyclinE decreased and phosphorylation of cdc2 Tyrl5 increased.For further exploration of BAP1 function in cell cycle regulation, EGFP-BAP1 was transfected into HeLa, A549 and BAP1 defective non-small lung carcinoma cell NCI-H226. It was observed that transfected cells became shrink and circle, losing its origin shape and their growth was inhibited. ThenIn general, in the present research, a novel protein BAP1 belonging to the deubiquitinanting enzymes was characterized from Artemia pathenogenetica. It was observed that depletion or overexpression of BAP1 in artemia or other cells lead to changes of cell cycle regulation. From these we proposed that BA1 participated in the G1/S transition in artemia, involving in the formation of the encyst embryos.