The Identification And Functional Study Of The Predicted RCC1Domain Of MARVELD1

MARVELD1is a new candidate tumor suppressor gene corresponding to theprotein of approximately18kDa，and its full-length of encoding mRNA is3238bp.Early detections suggest in a variety of tumor cells or tissues low MARVELD1expression level phenomenon exists. Functional studies show that MARVELD1playsimportant role in the regulation of cell cycle, cell proliferation, cell invasion andmigration, apoptosis, DNA damage repair process and so on. RCC1(regulator ofchromosome condensation-1), is reported to be the only one cell RanGEF (RanGuanine Exchange Factor) molecule, which catalyzes the formation of RanGTP, it isalso critical in functions of cell proliferation, cell cycle regulation, spindle formation,DNA replication, and the process of remodeling the nuclear envelope. Domainprediction analysis showed MARVELD1has a RCC1homology domain, so our studycame to explore the interactions of the function between MARVELD1and RCC1.Firstly, immunofluorescence assay showed that MARVELD1co-localization withRan in cells, while there may be weak co-localization between MARVELD1and RCC1.Furthermore, using of RCC1homology domain deletion truncated proteinMARVELD1-RCC1, we discovered the co-localization disappeared, indicating that thisdomain was necessary for MARVELD1to interact with Ran. Thenco-immunoprecipitation assay demonstrated a strong interaction between MARVELD1and RanGTP, otherwise a weak one for MARVELD1and RCC1. Moreover, weconstructed a recombinant plasmid pGEX-6P-1/RanBP1, and confirmed the interactionthrough GST-pull down assay.Next, we analyzed RCC1and Ran expression in cells with low or highMARVELD1expression level, and it displayed a positive correlation regulation forMARVELD1and RCC1expression, but had no difference on the expression of Ran.RNAi technology was carried out to inhibit MARVELD1, RCC1and Ran expression,and then the cells were staining by DAPI. The results showed that inhibition ofMARVELD1, RCC1or Ran caused mitosis abnormality, and knockdown of Ran led toa large number of cell death, suggesting MARVELD1may be involved in the regulationof mitosis. Next the statistics of DAPI fluorescence intensity promped MARVELD1andRCC1likely had similarities on chromatin condensation.In conclution, our study proved the interaction between MARVELD1and RanGTP,and comfirmed the existence of the predicted RCC1domain.