Nuclear PRP20 protein is required for mRNA export.

The yeast PRP20 protein is highly homologous in structure and function to the RCC1 protein of higher eukaryotes. The RCC1 protein is involved in the regulation of the onset of mitosis, whereas the PRP20 protein was shown to be required for accurate and efficient mRNA metabolism. The first observable phenotype in mutant prp20 cells when shifted from permissive to non-permissive temperature is a loss of nuclear PRP20 protein. Concomitantly, an accumulation of poly(A)+ RNA in the nucleus is observed. The temperature-sensitive RCC1 allele in the mutant hamster cell line tsBN2 leads to a similar accumulation of mRNA in the nucleus.     

The maternally expressed Drosophila gene encoding the chromatin-binding protein BJ1 is a homolog of the vertebrate gene Regulator of Chromatin Condensation, RCC1.

Using monoclonal antibodies I have identified a nuclear protein of Drosophila, BJ1 (Mr approximately 68 kd), and isolated its gene. Biochemical analysis demonstrates that the BJ1 protein is associated with nucleosomes and is released from chromatin by agents which intercalate into DNA, as previously shown for the high mobility group proteins (HMGs). On polytene chromosomes the protein is localized in all bands, with no preference for particular loci. Both the BJ1 protein and in particular the BJ1 mRNA are strongly expressed maternally. In early embryos all nuclei contain equal amounts of BJ1. During neuroblast formation, BJ1 mRNA becomes restricted to cells of the central nervous system, and higher protein levels are found in the nuclei of this tissue. In late embryonic stages, the mRNA almost completely disappears, but significant amounts of BJ1 protein persist until morphogenesis. The BJ1 gene encodes a 547 amino acid polypeptide featuring two different types of internal repeats. The sequence from amino acids 46 to 417 containing seven repeats of the first type has been highly conserved in evolution. 45% of the amino acids in this region are conserved in seven similar tandem repeats of the human gene Regulator of Chromatin Condensation, RCC1. The phenotype of a cell line carrying a mutation of RCC1 suggested a main function for this gene in cell cycle control. A yeast gene, SRM1/PRP20, also contains these repeats and shows 30% amino acid identity to BJ1 in this region. Mutations in this gene perturb mRNA metabolism, disrupt nuclear structure and alter the signal transduction pathway for the mating pheromone. Complementation experiments argue for a common function of these genes in the different species. I propose that their gene products bind to the chromatin to establish or maintain a proper higher order structure as a prerequisite for a regulated gene expression. Disruption of this structure could cause both mis-expression and default repression of genes, which might explain the pleiotropic phenotypes of the mutants.     

Overexpression of Yeast Homologs of the Mammalian Checkpoint Gene Rcc1 Suppresses the Class of α-Tubulin Mutations That Arrest with Excess Microtubules

Microtubules in eukaryotic cells participate in a variety of nuclear and cytoplasmic structures, reflecting functional requirements and cell cycle position. We are studying the cellular regulation of microtubule assembly and organization in the yeast Saccharomyces cerevisiae. We screened for genes that when overexpressed suppress the growth phenotype of conditional mutants in α-tubulin that arrest with excess microtubules at the nonpermissive temperature (class 2 mutations). Here we describe one such suppressing element, called ATS1 (for Alpha Tubulin Suppressor). Overexpression of this gene rescues both the growth and microtubule phenotypes of all class 2 mutations, but not the cold-sensitive mutations that arrest with no microtubules (class 1 mutations). Deletion of ATS1 confers a modest slow growth phenotype which is slightly enhanced in strains containing both a deletion of ATS1 and a class 2 tub1 mutation. The predicted ATS1 protein contains 333 amino acids and has considerable structural homology to the products of both the mammalian mitotic control gene RCC1 and the S. cerevisiae gene SRM1/PRP20. Overexpression of SRM1/PRP20 also suppresses class 2 mutants. The results suggest that this family of genes may participate in regulatory interactions between microtubules and the cell cycle.     

Premature chromosome condensation is induced by a point mutation in the hamster RCC1 gene.

At the nonpermissive temperature, premature chromosome condensation (PCC) occurs in tsBN2 cells derived from the BHK cell line, which can be converted to the Ts+ phenotype by the human RCC1 gene. To prove that the RCC1 gene is the mutant gene in tsBN2 cells, which have RCC1 mRNA and protein of the same sizes as those of BHK cells, RCC1 cDNAs were isolated from BHK and tsBN2 cells and sequenced to search for mutations. The hamster (BHK) RCC1 cDNA encodes a protein of 421 amino acids homologous to the human RCC1 protein. In a comparison of the base sequences of BHK and BN2 RCC1 cDNAs, a single base change, cytosine to thymine (serine to phenylalanine), was found in the 256th codon of BN2 RCC1 cDNA. The same transition was verified in the RCC1 genomic DNA by the polymerase chain reaction method. BHK RCC1 cDNA, but not tsBN2 RCC1 cDNA, complemented the tsBN2 mutation, although both have the same amino acid sequence except for one amino acid at the 256th codon. This amino acid change, serine to phenylalanine, was estimated to cause a profound structural change in the RCC1 protein.     

Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily.

The RCC1 gene of mammals encodes a guanine nucleotide release protein (GNRP). RCC1 and a homolog in Saccharomyces cerevisiae (MTR1/PRP20/SRM1) have previously been implicated in control of mRNA metabolism and export from the nucleus. We here demonstrate that a temperature-sensitive fission yeast mutant which has a mutation in a homologous gene, and two of three additional (mtr1/prp20/srm1) mutants accumulate nuclear poly(A)+ RNA at 37 degrees C. In S.cerevisiae, maturation of rRNA and tRNA is also inhibited at 37 degrees C. Nevertheless, studies with the corresponding BHK-21 cell mutant indicate that protein import into the nucleus continues. MTR1 homologs regulate RNA processing at a point which is distinct from their regulation of chromosome condensation since: (i) poly(A)+ RNA accumulation in the fission yeast mutant precedes chromosome condensation, and (ii) unlike chromosome condensation, accumulation of nuclear poly(A)+ RNA does not require p34cdc28 kinase activation or protein synthesis. Moreover, experiments involving inhibition of DNA synthesis indicate that the S.cerevisiae homolog does not govern cell cycle checkpoint control. Since RCC1p acts as GNRP for Ran, a small nuclear GTPase of the ras superfamily, we have identified two homologs of Ran in S.cerevisiae (CNR1 and CNR2). Only CNR1 is essential, but both code for proteins extremely similar to Ran and can suppress mtr1 mutations in allele-specific fashion. Thus, MTR1 and its homologs appear to act as GNRPs for a family of conserved GTPases in controlling RNA metabolism and transport. Their role in governing checkpoint control appears to be restricted to higher eukaryotes.     

Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis.

The temperature-sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCC1 gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non-permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the non-permissive temperature, p34cdc2 was dephosphorylated and M-phase specific histone H1 kinase was activated. However, in G1 phase, shifting to the non-permissive temperature did not activate p34cdc2 histone H1 kinase. The activation of p34cdc2 histone H1 kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone H1 kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase-microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone H1 kinase activity. The kinetics of p34cdc2 histone H1 kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1.     

Allelism of PSO4 and PRP19 links pre-mRNA processing with recombination and error-prone DNA repair in Saccharomyces cerevisiae.

The radiation-sensitive mutant pso4-1 of Saccharomyces cerevisiae shows a pleiotropic phenotype, including sensitivity to DNA cross-linking agents, nearly blocked sporulation and reduced mutability. We have cloned the putative yeast DNA repair gene PSO4 from a genomic library by complementation of the blocked UV-induced mutagenesis and of sporulation in diploids homozygous for pso4-1. Sequence analysis revealed that gene PSO4 consists of 1512 bp located upstream of UBI4 on chromosome XII and encodes a putative protein of 56.7 kDa. PSO4 is allelic to PRP19, a gene encoding a spliceosome-associated protein, but shares no significant homology with other yeast genes. Gene disruption with a destroyed reading frame of our PSO4 clone resulted in death of haploid cells, confirming the finding that PSO4/PRP19 is an essential gene. Thus, PSO4 is the third essential DNA repair gene found in the yeast S.cerevisiae.     

Apoptosis Is Induced in BHK Cells by the tsBN462/13 Mutation in the CCG1/TAFII250 Subunit of the TFIID Basal Transcription Factor

A temperature-sensitive (ts) mutant of the BHK21 cell line derived from golden hamsters, tsBN462 has a mutation in the gene encoding the largest subunit of the TFIID complex, TAF_II250/p230/CCG1, and arrests in the G1 phase at the nonpermissive temperature, 39.5°C. We found that tsBN462 cells underwent apoptosis following growth arrest at 39.5°C, suggesting a role for CCG1 as a repressor of apoptosis. By electron microscopic observation, tsBN462 cells at 39.5°C showed characteristic features of apoptosis. Apoptosis was not suppressed by expression of Bc1-2 or the adenovirus E1B 19 kDa protein. Cell death was suppressed completely by expression of wild-type CCG1 and partially by wild-type p53, a growth suppressor protein. Cell cycle arrest induced by p53 may help survival of tsBN462 cells at 39.5°C. Apoptosis was accelerated in SV40 large T antigen-transformed tsBN462 cells at 39.5°C where SV40 large T antigen formed a complex with p53, implying that the apoptosis of tsBN462 cells at 39.5°C occurred in a p53-independent manner. Our results suggest that CCG1/TAF_II250 is required for the expression of factors regulating apoptosis.     

RanBP1, a Ras-like nuclear G protein binding to Ran/TC4, inhibits RCC1 via Ran/TC4

A human protein that is 92% identical and 97% homologous at the amino acid level to RanBP1 from mouse was identified by the two-hybrid method, using two types of target cDNAs fused to sequences encoding the GAL4 DNA-binding domain. The target cDNAs encoded the human Ran/TC4 and human RCC1 proteins, respectively. An in vitro binding experiment showed that RanBP1 binds to RCC1 with the aid of Ran. Partially purified, GST-fused RanBP1 inhibited RCC1-stimulated guanine nucleotide release from Ran in vitro. Consistent with this in vitro finding, overproduction of human RanBP1 was detrimental to growth of tsBN2, a temperature-sensitive BHK21 hamster cell line defective in the RCC1 gene, and inhibited the growth of the Saccharomyces cerevisiae rcc1 mutants prp20, mtr1 and srm1. The specific effect of RanBP1 on rcc1 ^– cells was confirmed by the finding that overproduction of RanBP1 induces significant levels of expression of a FUS1-lacZ gene and an increase in mating efficiencies in a ste3, pheromone receptor-deficient yeast mutant. This phenotype is similar to the srm1, a mutant isolated as a suppressor that restores mating to receptorless mutants. These findings indicate that RanBP1 negatively regulates RCC1.     

Molecular cloning of the cDNA of human X chromosomal gene (CCG1) which complements the temperature-sensitive G1 mutants, tsBN462 and ts13, of the BHK cell line.

The tsBN462 cell line, a temperature-sensitive (ts) mutant isolated from the hamster cell line, BHK21/13 has a ts defect in G1 progression and belongs to the same complementation group as the ts13 cell line. We cloned human cDNA which can complement both tsBN462 and ts13 mutations, from the cDNA library of the secondary ts+ transformant (K-1-1) of tsBN462 cells using, as a probe, the isolated human X chromosomal genomic DNA. The cloned DNA is 5.3 kb long and has an open reading frame of 4662 bp, encoding a protein of 178,768 daltons. The putative protein is hydrophilic with a tandem repeat of 120 amino acids in the C-terminal region. An amino acid sequence (PPKKKRRV), similar to the consensus sequence for the nuclear translocation signal, is located immediately before the tandem repeat of amino acids.     

dad-1, an endogenous programmed cell death suppressor in Caenorhabditis elegans and vertebrates.

Programmed cell death (apoptosis) is a normally occurring process used to eliminate unnecessary or potentially harmful cells in multicellular organisms. Recent studies demonstrate that the molecular control of this process is conserved phylogenetically in animals. The dad-1 gene, which encodes a novel 113 amino acid protein, was originally identified in a mutant hamster cell line (tsBN7) that undergoes apoptosis at restrictive temperature. We have identified a dad-1 homologue in Caenorhabditis elegans (Ce-dad-1) whose predicted product is > 60% identical to vertebrate DAD-1. A search of the sequence databases indicated that DAD-1-like proteins are also expressed in two plant species. Expression of either human dad-1 or Ce-dad-1 under control of a C.elegans heat-shock-inducible promoter resulted in a reduction in the number of programmed cell death corpses visible in C.elegans embryos. Extra surviving cells were present in these animals, indicating that both the human and C.elegans dad-1 genes can suppress developmentally programmed cell death. Ce-dad-1 was found to rescue mutant tsBN7 hamster cells from apoptotic death as efficiently as the vertebrate genes. These results suggest that dad-1, which is necessary for cell survival in a mammalian cell line, is sufficient to suppress some programmed cell death in C.elegans.     

Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation.

The tsBN2 cell line, a temperature-sensitive (ts) mutant of baby hamster kidney cell line BHK21/13, seems to possess a mutation in the gene that controls initiation of chromosome condensation. At the nonpermissive temperature (39.5 degrees C), the chromatin of tsBN2 cells is prematurely condensed, and the cells die. Using tsBN2 cells as a recipient of DNA-mediated gene transfer, we investigated a human gene that is responsible for regulation of chromosome condensation and cell proliferation. We found that the human gene complementing the tsBN2 mutation resides in the area of the 40- to 50-kilobase HindIII fragment, derived from HeLa cells. Based on this finding, we initiated cloning of a human gene complementing the tsBN2 mutation. From lambda and cosmid libraries carrying partial digests of DNA from the secondary transformants, the 41.8-kilobase HindIII fragment containing the human DNA was isolated. The cloned human DNA was conserved in ts+ transformants through primary and secondary transfections. Two cosmid clones convert the ts- phenotype of tsBN2 cells to ts+ with more than 100 times a higher efficiency, compared with cases of transfection with total human DNA. Thus, the cloned DNA fragments contain an active human gene that complements the tsBN2 mutation.     

Activation of Cdh1-dependent APC is required for G1 cell cycle arrest and DNA damage-induced G2 checkpoint in vertebrate cells.

Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (Cdh1-APC) activity targets mitotic cyclins from the end of mitosis to the G1 phase. To investigate the function of Cdh1 in vertebrate cells, we generated clones of chicken DT40 cells disrupted in their Cdh1 loci. Cdh1 was dispensable for viability and cell cycle progression. However, similarly to yeast and Drosophila, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27(Kip1). Further more, we found that Cdh1(-/-) cells fail to maintain DNA damage-induced G2 arrest and that Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest.