Search

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.

Supplementary Figure 2 from Ionizing Radiation Induces Frequent Translocations with Delayed Replication and Condensation

Supplementary Figure 1 from Ionizing Radiation Induces Frequent Translocations with Delayed Replication and Condensation

Supplementary Figure 4 from Ionizing Radiation Induces Frequent Translocations with Delayed Replication and Condensation

Supplementary Figure 3 from Ionizing Radiation Induces Frequent Translocations with Delayed Replication and Condensation

OBJECTIVES Within the past several years, innovative communication tools have been established as viable quality improvement mechanisms in health care. Meanwhile, the economic and societal burdens of hospital-acquired conditions (HACs) continue to rise. Various interventions have been attempted to reduce HACs. This study evaluated the effectiveness of a communication tool in reducing risk factors for HACs. METHODS A communication tool aimed at reducing HAC risk factors was developed by an interdisciplinary team of physicians and nurses and tested in a simple before-after quality improvement study. It included 8 components: ambulation/fall risk, blood glucose greater than 200 mg/dL, central venous catheters, deep venous thrombosis prophylaxis, erosions of the skin/dermal ulcers, Foley/urinary catheters, got communication, and heart monitor/telemetry. This communication tool facilitated multidisciplinary communication. The nurses completed it nightly, and the physicians reviewed the communication tool each morning and, when appropriate, addressed components of care that were out of compliance with best practices. RESULTS The use of the ABCs of Hospitalized Patients Communication Tool led to daily improvements with reduction in the percentage of patients with blood glucose greater than 200 mg/dL from 43.3% to 35.1%, reduction in the use of central venous catheters from 8.2% to 1.0% of patients, increase in the use of chemical deep venous thrombosis prophylaxis from 45.4% of patients to 56.7%, reduction in the use of urinary catheters from 27.6% to 13.2%, and decrease in use of telemetry from 67.5% to 55.1%. All of the results have P < 0.05. These improvements were sustained over time. CONCLUSIONS Implementation of a multidisciplinary communication tool serves as a simple, resource-conscious, and customizable instrument to reduce the risk factors for developing HACs. This communication tool can be easily disseminated and used by other institutions.

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.

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.

The generation of functional mRNA in eukaryotes requires the accurate removal of noncoding regions (introns) from pre-mRNA by a process termed pre-mRNA splicing (14, 25). Splicing takes place in the spliceosome, a dynamic RNA-protein complex that assembles in a stepwise manner on the pre-mRNA (10, 14). The spliceosome is composed of small nuclear ribonucleoprotein particles (snRNPs) and extrinsic (non-snRNP) factors. The recognition of exon/intron boundaries, the splice sites, by the splicing apparatus is a critical step in processing of both constitutively and alternatively spliced pre-mRNAs. U1 snRNP defines the 5′ splice site, and U2 snRNP defines the branchpoint sequence (3, 10, 14, 17). Since in most cases the first AG dinucleotide downstream of the branchpoint is used as the 3′ splice site, by defining the branchpoint, U2 snRNP defines the 3′ splice site (18, 27). Targeting of U2 snRNP to the branch site requires the extrinsic splicing factor U2 snRNP auxiliary factor (U2AF). U2AF binds site specifically to the intron pyrimidine tract between the branchpoint sequence and 3′ splice site at an early step in spliceosome assembly and recruits U2 snRNP to the branch site (22, 29, 42). Regulation of 3′ splice site choice, both positive and negative, can be realized by influencing the pyrimidine tract binding of U2AF (33, 35, 45). Because U2AF is a major determinant in 3′ splice site selection, it has been the subject of extensive biochemical and genetic investigation. Human U2AF is a heterodimer composed of a 65-kDa large subunit (hU2AF65) and a 35-kDa small subunit (hU2AF35) (41). Both subunits are conserved in other organisms (40), and U2AF homologs have been identified in Drosophila melanogaster (9, 21), Schizosaccharomyces pombe (16, 36), and Caenorhabditis elegans (3a, 39). The Drosophila U2AF large (dU2AF50)- and small (dU2AF38)-subunit homologs are 50 and 38 kDa, respectively (9, 21). The U2AF large subunit contains three RNA recognition motifs (RRMs) and an amino-terminal arginine-serine-rich (RS) domain (42). The small subunit contains a highly degenerate RRM (pseudo-RRM) (2), two Zn2+ binding motifs (37), and a carboxyl-terminal RS domain and glycine-rich region (43). Both U2AF subunits are involved in recognition of the intron pyrimidine tract. The large subunit (hU2AF65 and dU2AF50) is required for site-specific pyrimidine tract binding (9, 42). The small subunit acts as a cofactor to stabilize the large subunit on the pyrimidine tract, apparently through protein-protein interactions with constitutive and alternative splicing factors (38, 45). While it has been firmly established that all three RRMs on hU2AF65 are necessary for high-affinity RNA binding, a role for the large-subunit RS domain in RNA binding remains unresolved (11, 42). In one study removal of the hU2AF65 RS domain had a modest effect on RNA binding (42). In a second study, the RS domain was found to be absolutely required for RNA binding (11). In vitro splicing assays using U2AF-depleted extracts prepared by two independent methods have identified independent and essential roles for the two U2AF RS domains: the large-subunit RS domain is required to target U2 snRNP to the branch site (34, 42), and in the immunodepleted extracts under certain conditions, the small-subunit RS domain is apparently necessary for protein-protein interactions with constitutive and alternative splicing factors to stabilize hU2AF65 on the pyrimidine tract (38, 45). In contrast to the essential roles assigned to the two U2AF RS domains in vitro, molecular genetic analysis of the Drosophila U2AF RS domains indicates that either one of the RS domains is dispensable in vivo (19). Importantly, at least one RS domain on U2AF is essential for viability (19). The observation that the dU2AF38 RS domain is not essential in vivo (19) refocused our attention on domains present in the U2AF small subunit that are phylogenetically conserved. In an exhaustive database search for proteins containing RRMs, some of the signature sequences of this motif were identified in hU2AF35 (2). These sequences are also present in the Drosophila and S. pombe small-subunit homologs (21, 36). Although some of the most conserved residues in the RNA recognition motif are present in the U2AF small subunits, the RNP-1 octamer is highly degenerate and the RNP-2 hexamer is absent. Since these defining submotifs and other conserved residues are not present in the U2AF small-subunit RRM, it was termed a degenerate RRM or pseudo-RRM (2). Degenerate RRMs have been identified in a collection of RNA binding proteins, including several of the SR proteins, the pyrimidine tract binding protein, and the large subunit of U2AF (10). The degenerate RRMs in SRp30a (ASF/SF2) (4, 46), pyrimidine tract-binding protein (15), and hU2AF65 (42) were all found to be required for high-affinity RNA binding. Two putative Zn2+ binding domains, one on either side of the pseudo-RRM, were recently identified in hU2AF35 in a database search (37). These Cys3His Zn2+ binding motifs are conserved in all three small subunit homologs. Though sequence-specific RNA binding has not been described for proteins that contain this type of Zn2+ binding motif, several proteins that have this domain are involved in RNA metabolism (37). The evolutionary conservation of the pseudo-RRM and the two Zn2+ binding motifs in all three U2AF small subunit homologs suggested to us that these domains are important for function. The lack of requirement for the dU2AF38 RS domain in vivo prompted us to search for novel biochemical activities associated with the small subunit. The phylogenetically conserved, degenerate RRM (2) and two Zn2+ binding motifs (37) in the small subunit suggested that it might participate in RNA binding. While we detected weak RNA binding activity for the Drosophila small subunit on its own, we found that when complexed with the large subunit, dU2AF pyrimidine tract binding affinity increased 20-fold. This increase in RNA binding activity was not specific to Drosophila U2AF; the human U2AF heterodimer bound RNA with 15-fold-higher affinity than hU2AF65. Surprisingly, removal of the dU2AF38 RS domain abolished the increase in binding activity of the dU2AF heterodimer, indicating that the RS domain is necessary for high-affinity binding. Deletion of the dU2AF50 RS domain (dU2AF50ΔRS) dramatically reduced RNA binding activity of the large-subunit monomer. High-affinity binding was restored when the dU2AF38 RS domain was supplied in trans to dU2AF50ΔRS. These data suggest that high-affinity RNA binding activity requires at least one RS domain on U2AF, which is consistent with the requirement for at least one RS domain in vivo.

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 (hU2AF65) and small (hU2AF35) subunits. Both subunits contain a domain enriched in arginine-serine dipeptide repeats termed an RS domain. The two U2AF RS domains have been assigned essential and independent roles in spliceosome assembly in vitro-the hU2AF65 RS domain is required to target U2 snRNP to the branch site and the hU2AF35 RS domain is necessary for protein-protein interactions with constitutive and alternative splicing factors. We have investigated the functional requirements for the RS domains on the Drosophila U2AF homolog in vivo. In sharp contrast to its essential role in U2 snRNP recruitment in vitro, the RS domain on the Drosophila large subunit homolog (dU2AF50) was completely dispensable in vivo. Prompted by this unexpected result, we analyzed the RS domain on the Drosophila small subunit homolog (dU2AF38). Despite its requirement for enhancer-dependent splicing activity in vitro, the dU2AF38 RS domain was also inessential in vivo. Finally, we have tested whether the Drosophila U2AF heterodimer requires any RS domain. Flies mutant for both the small and large subunits could not be rescued by dU2AF50deltaRS and dU2AF38deltaRS transgenes. Therefore, in contrast to the separate roles assigned to the U2AF RS domains in vitro, our genetic data suggest that they may have redundant functions in vivo.

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 (dU2AF50) that interacts with the Drosophila small subunit (dU2AF38). A 28-amino-acid fragment on dU2AF50 that is both necessary and sufficient for interaction with dU2AF38 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 dU2AF50 allele. A mutation that completely abolished interaction with dU2AF38 was incapable of complementation, whereas dU2AF50 mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF50 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 dU2AF50 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.

The essential eukaryotic pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is required to specify the 3' splice 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.