Characterization And Functional Analysis Of Regulator Of Chromosome Condensation 1 From Tetrahymena Thermophila

Ran （Ras-like nuclear protein） is a small GTPase protein, is highly conserved in eukaryotes, belongs to the small G proteins family. Ran protein interaction with different regulatory factors mainly involved in the nucleocytoplasmic transport, spindle assembly, nuclear membrane reconstruction, DNA replication and cell cycle regulation. With auxiliary proteins, Ran performs the cycling change between RanGTP and RanGDP.RCC1 （regulator of chromosome condensation 1） is the only known Ran guanine nucleotide exchange factor, contains a conserved RLD （RCC1-like domain） domain, one side combined with Ran Protein, promote Ran-GDP into RanGTP and nucleocytoplasmic transport. RCC1 plays an important role in the microtubule assemblely, cell cycle regulation and telomeric silencing.Tetrahymena thermophila is a single-cellular the ciliated protozoa and has nuclear dimorphism, The somatic macronucleus is polyploid and the germline micronucleus is diploid. In addition, T. thermophila has two different reproduction type, asexual reproduction and sexual reproduction. In the asexual reproduction period, the micronuclei perform mitosis and the macronuclei perform amitosis. In the sexual reproduction stage, the micronuclei perform meiosis and mitosis; the parent macronucleus is degraded. The sophisticated nuclear structure of T. thermophila, is a good model system to study the structure and function of the nucleus. In our previous study, we found that Ran and RanBP1 play a vital role in the macronuclear amitosis, Furthermore, the abnormal expression of the two proteins affects cell proliferation. But as we know, at present, there are not reports for the function research of RCC1 in ciliate.In this study, we first identify a new RCC1 from the genome database of T. thermophila. Then,we analyzed the function of RCC1, The main results are as follows:1. Bioinformatic analysis of RCC1. By Blastp the Tetrahymena macronuclear genome databse （http://www.ciliate.org）, we identify the human RCC1 homo log, TTHERM₀0530380 gene. The gene from T.thermophila was 2541 bp, open reading frame is 2181 bp, predicted encoded 726 amino acids, the gene named Tetrahymena RCC1. Real-time RT-PCR showed that the RCC1 express in vegetative growth, starvation and conjugation stage in T. thermophila. In the conjugation stage, RCC1 showed higher expression levels. The result is consistent with the RCC1 expression profiling from Tetrahymena Microarray Expression Database （http://tfgd.ihb.ac.cn）. Bioinformatics analysis of RCC1 revealed that it contains a conserved RLD domain, and the evolution analysis of different RCC1 showed it is consistant with the species evolution.2. Immunofluorescence localization of HA-RCC1. pXS75-RCC1 containing a HA-tag was constructed and transformed into wild-type T. thermophila cells. The mutant cells were screened and identified by paromomycin resistance and PCR amplification. Immunofluorescence localization analysis showed that RCC1 specifically located in the MAC and MIC in the vegetative growth stage and starvation stage, HA-RCC1 localized in the parent macronucleus, meiotic micronucle and new macronuclei in conjugation stage. The RCC1 is nuclear specific functional protein.3. Over-expressed RCC1 affect the nuclear division. pXS75-RCC1 was constructed and transformed into wild-type T. thermophila. Endogenous MTT1 genes was replaced by RCC1 gene via homologous recombination with Cd2+induction and paromomycin resistance. The nuclei were stained with DAPI. The results showed that over-expressed RCC1 leads to the macronuclear abnormal division, and produced abnormal progeny cells.4. Knockdown RCC1 gene results in abnormal nuclear division. pNeo4-RCC1 was constructed and transformed into wild-type T. thermophila. Endogenous RCCl gene was replaced by Neo4 gene with homologous recombination. The nuclei were stained with DAPI. The results showed that knockdown RCC1 leads to the multiple micronucle, affect the normal development of the cell.In the study, we identify a new RCC1 gene from the T. thermophila, the abnormal expression of RCC1 influence the normal mitosis, this abnormal phenotype is consistent with the Ran mutants. RanGTPase signaling pathway is important for the cellular proliferation and the normal development of nuclei. RCC1 could take part in the RanGTPase signaling pathway, and regulate the nuclear development. RCC1 normal expression level plays a vital role in T. thermophila.