Certain chromosome rearrangements, found in cancer cells or in cells exposed to ionizing radiation, exhibit a chromosome-wide delay in replication timing (DRT) that is associated with a delay in mitotic chromosome condensation (DMC). We have developed a chromosome engineering strategy that allows the generation of chromosomes with this DRT/DMC phenotype. We found that approximately 10% of inter-chromosomal translocations induced by two distinct mechanisms, site-specific recombination mediated by Cre or non-homologous end joining of DNA double-strand breaks induced by I-Sce1, result in DRT/DMC. Furthermore, on certain balanced translocations only one of the derivative chromosomes displays the phenotype. Finally, we show that the engineered DRT/DMC chromosomes acquire gross chromosomal rearrangements at an increased rate when compared with non-DRT/DMC chromosomes. These results indicate that the DRT/DMC phenotype is not the result of a stochastic process that could occur at any translocation breakpoint or as an epigenetic response to chromosome damage. Instead, our data indicate that the replication timing of certain derivative chromosomes is regulated by a cis-acting mechanism that delays both initiation and completion of DNA synthesis along the entire length of the chromosome. Because chromosomes with DRT/DMC are common in tumor cells and in cells exposed to ionizing radiation, we propose that DRT/DMC represents a common mechanism responsible for the genomic instability found in cancer cells and for the persistent chromosomal instability associated with cells exposed to ionizing radiation.
The essential eukaryotic pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is required to specify the 3' splice site at an early step in spliceosome assembly. U2AF binds site-specifically to the intron polypyrimidine tract and recruits U2 small nuclear ribonucleoprotein to the branch site. Human U2AF (hU2AF) is a heterodimer composed of a large (hU2AF65) and small (hU2AF35) subunit. Although these proteins associate in a tight complex, the biochemical requirement for U2AF activity can be satisfied solely by the large subunit. The requirement for the small subunit in splicing has remained enigmatic. No biochemical activity has been found for hU2AF35 and it has been implicated in splicing only indirectly by its interaction with known splicing factors. In the absence of a biochemical assay, we have taken a genetic approach to investigate the function of the small subunit in the fruit fly Drosophila melanogaster. A cDNA clone encoding the small subunit of Drosophila U2AF (dU2AF38) has been isolated and sequenced. The dU2AF38 protein is highly homologous to hU2AF35 containing a conserved central arginine-and serine-rich (RS) domain. A recessive P-element insertion mutation affecting dU2AF38 causes a reduction in viability and fertility and morphological bristle defects. Consistent with a general role in splicing, a null allele of dU2AF38 is fully penetrant recessive lethal, like null alleles of the Drosophila U2AF large subunit.Generation of functional mRNA in eukaryotes requires the removal of noncoding regions (introns) from pre-mRNA by a process termed RNA splicing (1-3). Pre-mRNA splicing takes place in the spliceosome, a dynamic RNA-protein complex that assembles in a stepwise ATP-dependent manner on the pre-mRNA (1-3). The spliceosome is composed of small nuclear ribonucleoprotein particles (snRNPs) and extrinsic (non-snRNP) protein factors. Studies with human cell (HeLa) nuclear splicing extracts have shown that targeting of U2 snRNP to the branch site on the pre-mRNA, an early step in spliceosome assembly, requires a protein factor called U2AF (U2 snRNP auxiliary factor) (4). U2AF binds site-specifically to the intron polypyrimidine tract located between the branch site and the 3' splice site of the pre-mRNA (5). U2AF recruits U2 snRNP to the branch site in the first ATP-dependent step in spliceosome assembly. Recruitment of U2 snRNP to the branch site defines the 3' splice site since in most cases the first AG dinucleotide downstream of the branch site is used as the 3' splice site (6). Thus, U2AF plays a critical role in 3' splice site selection.Human U2AF (hU2AF) consists of two polypeptides, a 65-kDa large subunit (hU2AF65) and a 35-kDa small subunit (hU2AF35) (7). hU2AF65 contains three carboxyl-terminal ribonucleoprotein consensus sequence (RNP-CS) domains that mediate RNA binding and an amino-terminal arginineand serine-rich (RS) domain (5). All domains are essential for splicing in vitro (5). hU2AF35 contains a central RS domain and a carboxyl-terminal glycine...
The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary factor), plays a critical role in 3 splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia coli copurification assay to map the domain on the Drosophila U2AF large subunit (dU2AF 50 ) that interacts with the Drosophila small subunit (dU2AF 38 ). A 28-amino-acid fragment on dU2AF 50 that is both necessary and sufficient for interaction with dU2AF 38 was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF50 point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF 50 allele. A mutation that completely abolished interaction with dU2AF 38 was incapable of complementation, whereas dU2AF 50 mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF 50 allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF 50 recessive lethal allele. These data indicate that Drosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro.Generation of functional mRNA in eukaryotes requires the removal of noncoding sequences (introns) from pre-mRNA by a process termed RNA splicing (17, 24). Pre-mRNA splicing takes place in the spliceosome, a dynamic RNA-protein complex that assembles in a stepwise, ATP-dependent manner on the pre-mRNA (13, 17). The spliceosome is composed of small nuclear ribonucleoprotein particles (snRNPs) and extrinsic (non-snRNP) protein factors. The earliest steps in spliceosome assembly involve the specification of the exon and intron boundaries by U1 and U2 snRNP. U1 snRNP binds the 5Ј splice site, and U2 snRNP binds the branch site sequence (13,17,19). Since in most cases the first AG dinucleotide downstream of the branch site is used as the 3Ј splice site, U2 snRNP defines the 3Ј splice site (20,27). The branch site sequence in metazoan introns is highly degenerate and is not sufficient for U2 snRNP recognition (22). Targeting of U2 snRNP to the branch site requires the extrinsic protein factor U2AF (U2 snRNP auxiliary factor). U2AF binds site specifically to the intron pyrimidine tract located between the branch point sequence and the 3Ј splice site and recruits U2 snRNP to the branch site at an early step in spliceosome assembly (22,36). Regulation of 3Ј splice site choice, both positive and negative, can be modulated by U2AF binding to the intron pyrimidine tract (3,6,17). Thus, U2AF is a major determinant in 3Ј splice site selection.
The pre-mRNA splicing factor U2AF (U2 snRNP auxiliary factor) has an essential role in 3 splice site selection. U2AF binds the intron pyrimidine tract between the branchpoint and the 3 splice site and recruits U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF The generation of functional mRNAs in eukaryotes requires the accurate removal of noncoding sequences (introns) from pre-mRNAs by a process termed pre-mRNA splicing (Moore et al. 1993;Sharp 1994;Kramer 1996). Pre-mRNA splicing takes place in the spliceosome, a dynamic RNA-protein complex composed of small nuclear ribonucleoprotein particles (snRNPs) and extrinsic (nonsnRNP) protein factors. The earliest steps in spliceosome assembly involve recognition of the 5Ј splice site by U1 snRNP and the branchpoint-3Ј splice site by U2 snRNP. Targeting of U2 snRNP to the branch site requires the extrinsic splicing factor U2AF (U2 snRNP auxiliary factor) (Ruskin et al. 1988). U2AF binds specifically to the intron pyrimidine tract located between the branchpoint and the 3Ј splice site and recruits U2 snRNP to the branch site at an early step in spliceosome assembly (Ruskin et al. 1988;Zamore et al. 1992;Staknis and Reed 1994). Regulation of 3Ј splice site choice, both positive and negative, can be realized by influencing the pyrimidine tract binding of U2AF (Tian and Maniatis 1993;Valcá rcel et al. 1993;Reed 1996).Human U2AF is a heterodimer composed of a 65-kD large subunit ( hU2AF 65 ) and a 35-kD small subunit (hU2AF 35
Certain chromosome rearrangements display a significant delay in replication timing that is associated with a delay in mitotic chromosome condensation. Chromosomes with delay in replication timing/delay in mitotic chromosome condensation participate in frequent secondary rearrangements, indicating that cells with delay in replication timing/delay in mitotic chromosome condensation display chromosomal instability. In this report, we show that exposing cell lines or primary blood lymphocytes to ionizing radiation results in chromosomes with the delay in replication timing/delay in mitotic chromosome condensation phenotype, and that the delay in replication timing/delay in mitotic chromosome condensation phenotype occurs predominantly on chromosome translocations. In addition, exposing mice to ionizing radiation also induces cells with delay in replication timing/delay in mitotic chromosome condensation chromosomes that persist for as long as 2 years. Cells containing delay in replication timing/delay in mitotic chromosome condensation chromosomes frequently display hyperdiploid karyotypes, indicating that delay in replication timing/delay in mitotic chromosome condensation is associated with aneuploidy. Finally, using a chromosome engineering strategy, we show that only a subset of chromosome translocations displays delay in replication timing/delay in mitotic chromosome condensation. Our results indicate that specific chromosome rearrangements result in the generation of the delay in replication timing/delay in mitotic chromosome condensation phenotype and that this phenotype occurs frequently in cells exposed to ionizing radiation both in vitro and in vivo.
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