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Mammalian Cells Have Two Functional RCC1 Proteins Produced by Alternative Splicing

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Mammalian Cells Have Two Functional RCC1 Proteins Produced by Alternative Splicing Journal of Cell Science 107, 2203-2208 (1994) 2203 Printed in Great Britain © The Company of Biologists Limited 1994 Mammalian cells have two functional RCC1 proteins produced by alternative splicing Junko Miyabashira, Takeshi Sekiguchi and Takeharu Nishimoto* Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812, Japan *Author for correspondence SUMMARY Previously we cloned two human RCC1 cDNAs that that had already been determined, and was found to be differed in their noncoding region. In this study, we have located between the 6th and 7th exons, designated as the 6′ found new human and hamster RCC1 cDNAs, which have exon. Both the 5′ and 3′ ends of the 6′ exon correspond to an even more different coding region from that of the pre- the GT-AG rules for splicing, indicating that human RCC1- viously cloned RCC1 cDNAs yet can complement the RCC1 I mRNAs are produced by alternative splicing. The finding mutation in the tsBN2 cell line. The newly found RCC1 that both humans and hamsters have the insertion at the cDNAs encode a protein (designated as RCC1-I) that has same RCC1 site suggests that the pattern of alternative an insertion of 31 (human) and 13 (hamster) amino acids splicing in the RCC1 gene has been conserved through at valine25 in the N-terminal region outside the RCC1- evolution. seven repeat. The inserted nucleotide sequence was searched for, within the human RCC1 genomic sequence Key words: RCC1, alternative splicing, tsBN2 INTRODUCTION required for coupling the completion of DNA replication with the initiation of mitosis. However, it remains unclear how the The RCC1 gene has been found to be mutated in the tsBN2 arrest in the G1 phase occurs. Interestingly, when tsBN2 cells cell line, which is a temperature-sensitive (ts) mutant of the synchronized by serum deprivation were allowed to grow by BHK21 cell line derived from golden hamsters (Uchida et al., the addition of serum at 39.5°C, the nonpermissive tempera- 1990), and to encode a very abundant chromosomal protein ture, both RNA synthesis and protein synthesis were greatly (Ohtsubo et al., 1989). RCC1 functions as a guanine inhibited (Nishimoto et al., 1981). This previous finding nucleotide-releasing protein on the Ran, which is a nuclear suggested to us that in tsBN2 cells, chromatin was condensed small Ras-like G protein (Bischoff and Ponstingl, 1991). even in the G1 phase at the nonpermissive temperature, Homologues of RCC1 have been cloned from hamster, resulting in an inhibition of total RNA synthesis and protein Xenopus (Nishitani et al., 1990), Drosophila (BJ1) (Frasch, synthesis. However, it is also possible that decondensation of 1991), S. cerevisiae (SRM1/PRP20/MTR1) (Clark et al., 1989; chromatin, which is carried out in the G1 phase in order to start Aebi et al., 1990; Kadowaki et al., 1993) and S. pombe (pim1- the cell cycle, was prevented at the nonpermissive temperature. d1+) (Matsumoto and Beach, 1991; Sazer and Nurse, 1994). Consistent with this notion, Sazer and Nurse (1994) suggested Except for pim1-d1+, these homologues have been reported to that a ts mutant of S. pombe, pim1-d1ts, which ceases to grow be functionally exchangeable (Ohtsubo et al., 1991; Fleis- at the end of mitosis, like tsBN2 cells, has a defect in chromatin chmann et al., 1991; Clark et al., 1991), indicating that RCC1 decondensation. pim1-d1ts, however, does not prematurely has been conserved structurally and functionally through enter mitosis. On the contrary, the RCC1-homologous gene of evolution (reviewed by Dasso, 1993). S. cerevisiae has been identified as a mutant that is defective A defect in the RCC1 gene leads to various phenotypes. In in either mRNA metabolism (prp20) (Aebi et al., 1990), or in tsBN2 cells, RCC1 disappears at the nonpermissive tempera- mRNA export from the nuclei (mtr1) (Kadowaki et al., 1993), ture. Upon loss of RCC1 function, tsBN2 cells undergoing and as a mutant that restores the mating capacity to receptor- DNA replication prematurely enter mitosis and are arrested at less mutants (srm1) (Clark and Sprague, 1989). Except for the end of mitosis, showing re-formed micronuclei that contain srm1, these mutants have no relation to the cell cycle. condensed chromatin (Nishitani et al., 1991). On the other However, prp20 and pim1-d1ts, in addition to tsBN2 cells, hand, exponentially growing tsBN2 cells cease to grow in G1 accumulate mRNA in their nuclei at the nonpermissive tem- phase at the nonpermissive temperature (Nishimoto et al., perature, indicating that these mutants have a defect in mRNA 1978). Premature initiation of mitosis suggests that RCC1 is export as does mtr1 (Kadowaki et al., 1993; Amberg et al., 2204 J. Miyabashira and others 1993). It is therefore logical that phenomena caused by a defect methods (Maniatis et al., 1989), and was then amplified using the in the RCC1 gene should reflect some aspects of RCC1 following synthetic nucleotides as primers: 5′ primer, GCCGACGT- function. GCACCAAGGACAGGAAG, and 3′ primer, AGCCAGGCCCTCA- pim1-d1ts can be suppressed by overexpression of the G GAGGCTTCATCA for KB cells; and 5′ primer, TCCCCGCAACG ′ protein spi1/fyt1, the S. pombe gene homologous to Ran AGGACAGGAAGATG, and 3 primer, ACTACTTCAGAAACAC- (Matsumoto and Beach, 1991; Sazer and Nurse, 1994). In CCGGACCGA for tsBN2 cells. The PCR products were subcloned into the pCRII vector (Invitrogen). addition, both prp20 and mtr1 can be suppressed by overex- pression of the G protein GSP/CNR, the S. cerevisiae gene Transformation of tsBN2 cells homologous to Ran (Belhumeur et al., 1993; Kadowaki et al., Amplified RCC1 cDNA was integrated into a mammalian expression − 1993). Suppression of these rcc1 mutations by the overex- vector, pcDEB∆ containing the hygromycin-resistant gene hygr pression of Ran-homologues indicates that Ran functions (kindly provided by Y. Nakabeppu, Kyushu University), and co-trans- downstream of RCC1 (reviewed by Dasso, 1993). The other fected into tsBN2 cells (2×105 cells/100 mm dish) with pSV2 neo mutant, srm1, however, cannot be suppressed by the overex- (200 ng/dish) by the calcium phosphate precipitation method pression of Ran homologues (Kadowaki et al., 1993). We described by Sekiguchi et al. (1988). After incubation at 33.5°C for two days, transfected cells were cultured either in the presence of reason that srm1 may have a defect in accepting a signal from r a position upstream of RCC1. Geneticin (Sigma) (0.8 mg/ml) at 33.5°C for selection of neo colonies, or in a normal medium at 39.5°C for selection of ts+ trans- To investigate the upstream and downstream pathways of formants. About two weeks later, surviving cells were fixed with the RCC1-Ran system, we began by isolating suppressors of formaldehyde and were stained with crystal violet. tsBN2 cells that overcome their temperature sensitivity by a In the case of double transfection with RCC1 and RCC1-I, the mutation outside the RCC1 gene. In this way, we found that RCC1 cDNA was first integrated into another mammalian expression animal cells have two functional RCC1 proteins produced by vector, pcDL-SRα296 (Takebe et al., 1988) and then transfected into alternative splicing. Previously, the human RCC1 gene was tsBN2 cells to isolate primary ts+ transformants. The RCC1-I cDNA found to have two promoters, resulting in two RCC1 mRNAs carried on the pcDEBD vector was then transfected into primary ts+ that have the same coding region (Ohtsubo et al., 1987; Furuno transformants, with the hygr colonies being isolated at 39.5°C. et al., 1991). Furthermore, our present results indicate that the expression of the RCC1 gene is subject to alternative splicing in the coding region. The pattern of alternative splicing of the RCC1 gene is the same for both human and hamster, suggest- RESULTS ing that the alternative splicing pattern of RCC1 genes is conserved through evolution. Identification of two RCC1 mRNAs differing in their coding regions Isolation of ts+ revertants from cultures of tsBN2 cells that can grow at the nonpermissive temperature was simply the starting MATERIALS AND METHODS point of this study. Our original purpose was to investigate the RCC1-Ran system by isolating ts+ revertants of tsBN2 cells Cell lines and culture conditions that have a mutation outside the RCC1 gene. Thus, in order to The tsBN2 cell line is a ts mutant of the BHK21 cell line derived from determine whether the original mutation of the RCC1 gene golden hamsters. The ts+ transformants of tsBN2 cells were con- exists in these ts+ revertants, we amplified the coding region of structed by transfecting the RCC1 and RCC1-I cDNAs as described + (Sekiguchi et al., 1988). All cell lines were cultured in Dulbecco’s the RCC1 mRNA expressed in ts revertants of tsBN2 cells. modified Eagle’s medium (DMEM) containing 10% calf serum and Amplified RCC1 cDNAs were subcloned into the pcRII vector maintained at 33.5°C (tsBN2), at 37.5°C (BHK21, KB), and at 39.5°C and then digested with EcoRI. Since the RCC1 cDNA has only (ts+ transformants of tsBN2 cells) in a humidified atmosphere con- one EcoRI site in the coding region, two EcoRI fragments, 0.4 taining 10% CO2. kb and 0.9 kb were expected to be produced by digestion with EcoRI. Immunoblotting analysis After complete digestion, about one fifth of the cDNA Cellular proteins electrophoresed on a 12.5% SDS-polyacrylamide gel clones was found to have EcoRI fragments longer than 0.9 kb, were transferred to a PVDF membrane (Immobilon-P, Millipore) in along with the 0.4 kb fragment. A representative result is transfer buffer (50 mM Tris, 40 mM glycine, 15% methanol and 0.02% SDS). Subsequently, the filter was blocked in blocking buffer shown in Fig.
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