Chromosome instability caused by mutations in the genes involved in transcription and splicing

ABSTRACT

Mutations in molecules involved in transcription and splicing can cause chromosome instability such as sister chromatid exchanges. We isolated and characterized responsible genes from mammalian temperature-sensitive mutant cells showing chromosome instability. A mutation in the largest subunit of RNA polymerase II affected DNA synthesis in S phase-arrested cells, resulting in abnormal induction of sister chromatid exchanges. The yeast mutant harboring a homologous mutation showed very similar phenotype to that of the mammalian mutant. A mutation in Smu1, which is involved in splicing, also affected DNA synthesis in S and G2 phase-arrested cells, resulting in abnormal induction of sister chromatid exchanges and chromosomal aberrations. These cells showed a connection between defects of RNA metabolism and induction of chromosome instability. Genome instability appeared to be caused by links between RNA metabolism and replication resulting in genomic recombination. RNA metabolism can be regarded as one possible driver of genome modification triggering genome evolution

Introduction

Transcription is the first step of gene expression carried out by RNA polymerase whereby a particular segment of DNA is copied into RNA (see [1] for comprehensive review). Three main types of such enzymes are present in eukaryotes: RNA polymerase I, II, and III. Active RNA polymerases II are concentrated within discrete factories wherein many different templates are transcribed simultaneously (for a book on the concept, see [1]). Polymerases are considered as being immobile (either fixed or engaged) in factories. Primary transcripts made by RNA polymerase II are modified by the addition of a 5′ cap and 3′ poly (A) tail and by the excision of internal sequences in a reaction known as splicing. As a result of this processing, primary transcripts in the nucleus are sent out to the cytoplasm as mature messages.

The DNA of all organisms must be duplicated before every cell division in a process called replication, by which individual strands in a DNA duplex are separated before each strand is copied into a new complementary partner (for reviews of the principles, see [1,2]). This process is carried out by many DNA polymerases, which replicate different parts of a chromosome simultaneously in large replication factories. DNA is unwound at specific sites in the genome called origins and new strands are synthesized to grow bi-directionally from these origins. Numerous proteins associated with the replication help in the initiation, elongation, and termination of DNA synthesis.

A replication fork consists of a group of proteins that influence the activity of DNA replication and is built by helicases (see [3] for a comprehensive review). These enzymes break the hydrogen bonds linking the two DNA strands together, thus producing two branches, each comprising a single strand of DNA. These two strands become the template for the leading and lagging strands of DNA replication. DNA polymerase correctly matches complementary nucleotides to the templates. Replication stress typically occurs during DNA replication and can result in a stalled replication fork (for comprehensive reviews, see [4–6]). It is induced by various endogenous and exogenous stresses, which include DNA damage, excessive compacting of chromatin, and overexpression of genes. Replication stress can lead to genome instability, ageing, and cancer [7,8].

Here, I summarize several of our studies to assess the chromosome instability caused by mutations in the genes involved in RNA metabolism. We originally isolated 25 temperature-sensitive (ts) mutants from the CHO-K1 Chinese hamster ovary cell line to determine the genes involved in maintaining chromosome integrity [9]. In two of these mutants, ts defects in two molecules involved in RNA metabolism, i.e. the largest subunit of RNA polymerase II (Rpb1) and Smu1, a protein involved in splicing, caused chromosome instability [10,11].

Transcription

Temperature-sensitive mutant cell tsTM4

RNA polymerase II is a multi-subunit enzyme that transcribes most genes in eukaryotes [12,13]. It creates enormous complexes that are associated with other proteins with functions related to splicing and to polyadenylation [14,15]. Rpb1 is the largest and is the catalytic subunit in the core enzyme. tsTM4, a ts mutant of CHO-K1 that grows at 34°C but not at 39°C, contains an amino acid substitution that is a proline replaced by a serine at position 1006 in Rpb1 (Table 1) [9,10]. This mutant showed a complex phenotype of abnormally induced sister chromatid exchange and decreased DNA synthesis when cells were arrested in the S phase at the nonpermissive temperature, 39°C (Table 1) [9]. The relative rates of ‘run-on’ transcription ([³²P]UTP incorporation measured by using permeabilized cells) at 34°C and 39°C were roughly similar for the wild-type CHO-K1 and ts mutant tsTM4 [16]. A significant decrease of ‘run-on’ transcription at 39°C was not detected by this measurement although it did not reflect the exact transcriptional activity of RNA polymerase II. The results suggested that the transcriptional activity appeared to be normal in mutant cells even at 39°C. The effects of the mutation only slowly become apparent on transcription at 39°C. Therefore, a ts mutation of RNA polymerase II found in tsTM4 cells might appear to impact the replication directly. However, genes involved in transcription are critical for protein expression, and the loss of their function is known to affect replication indirectly and directly. It is undeniable that indirect effects of transcription on replication result in defects in the expression of molecules that safeguard the genome.

Table 1.

Two temperature-sensitive CHO-K1 mutant cell lines showing chromosome instability.

Name	DNA synthesisa	Cell cycle arrest	Induction of SCEsb	Responsible gene	References
tsTM4	Decrease	S	Yes	Rpb1: The largest subunit of RNA polymerase II	9, 10
tsTM18	Decrease	S and G2	Yes	Smu1: A protein involved in spliceosomes	9, 11

^aActivity at 39°C

^bSCEs: sister chromatid exchanges

Yeast mutant harboring homologous mutation with tsTM4 cells

To confirm ts mutation in tsTM4 and for genetic analysis using yeast, we isolated a mutant of Schizosaccharomyces pombe that substitutes the homologous proline residue with a serine. Growth of the mutant haploid cells was delayed at the nonpermissive temperature of 37°C [17]. Tetrad analysis showed these phenotypes to be associated exactly with the mutation [17]. In diploid mutant cells, chromosome instability was found based on the loss of intragenic complementation between two alleles of the ade6 gene [17]. In addition, FACS analysis revealed an abnormal fraction of cells with intermediate DNA content [17]. This fraction may reflect either numerous cells in the S phase or cells with abnormal DNA content caused by chromosome instability, suggesting that this yeast mutant shows a similar phenotype to that of the mammalian mutant, tsTM4.

In a later study, RNA polymerase II mutants in another yeast, Saccharomyces cerevisiae, showed an increase in genetic instability as detected by hyper-recombination, sensitivity to DNA damage, and dependency on double-strand break repair functions for viability, again suggesting a link between the mutations in RNA polymerase II and genome instability [18].

Conflict between transcription and replication

Transcription and replication are considered to be two major motor apparatuses acting on genome DNA that are shared as a template for both. Each forms large complexes known as factories, which comprise many factors related to transcription and replication (see [1] for a comprehensive review). Both are regulated not to obstruct or hinder each other in usual growth conditions. However, when they become uncontrollable, they may cause a head-on collision, a rear-end collision, or dragging. Figure 1 presents a model of the tug of war between transcription and replication. Although accidents are usually avoided, they may sometimes be caused intentionally or systematically (see [19] for a comprehensive review).

Figure 1.

Model for a tug of war between transcription and replication. Transcription and replication are considered to be two motor apparatuses acting on genome DNA that are shared as a template for both. Red and blue arrowheads respectively indicate the direction of the two apparatuses of transcription and replication (leading strand), and the red and blue arrows indicate the respective pulling forces of transcription and replication. They are regulated not to obstruct or hinder each other in the usual nucleus, but if this regulation becomes uncontrollable, they may cause a head-on collision, a rear-end collision, or dragging. These accidents may sometimes be caused intentionally or systemically.

Gene evolution is promoted by conflict between replication and transcription

Lagging-strand replication was suggested to shape the mutational landscape of the genome [20]. Before this report, Paul and colleagues showed that bacterial genes in the lagging strand of replication undergo higher rates of mutation and more rapid adaptive evolution than those in the leading strand [21]. These results suggest that increased conflict between replication and transcription machineries is at least partially responsible for the higher rates of mutagenesis in genes encoded within the lagging strand, thus promoting faster adaptive evolution of core genes in bacteria. Interestingly, they found that head-on conflicts between replication and transcription can result in more mutagenesis than co-directional conflicts and that adaptive structural variation in the coded proteins is significantly increased by these encounters.

Replication, transcription, and recombination in ribosomal RNA gene

The structure of the ribosomal RNA gene (rDNA) is highly repetitive and highly conserved from yeast cells to human cells. Although recombination between the repeated rDNA results in the loss of copies that is detrimental to cells, a unique gene amplification system in the rDNA repeats helps to restore the copy number (see [22] for a comprehensive review). Transcription in this region functions in this gene amplification. A non-coding promoter located in the intergenic spacer of rDNA induces unequal sister chromatid recombination allowing amplification to recover the copy number. Sir2, a histone deacetylase, can repress this intergenic transcription to maintain the repeat number at an appropriate level.

The replication fork barrier (RFB) site is also a unique element in rDNA (see the review [22] for details). The RFB helps to avoid collisions between the replication and transcription machineries because rDNA is the most actively transcribed region in the genome. Fob1 is an essential protein for rDNA maintenance that specifically associates with the RFB sequence and regulates the number of repeats.

Amplification of DHFR gene in mammalian cells

An increase in the gene copy number promotes the divergence of the copies, which is critical to the process of creating novel genes for evolution. Gene amplification is one of the recombination events occurring in somatic cells to increase the gene copy number. The most well-known gene amplification in mammalian cells is that occurring in the dihydrofolate reductase (DHFR) gene by treatment with methotrexate (MTX), which competitively inhibits DHFR protein [23]. MTX is used in cancer therapy, and MTX-resistant cancer cells appear as a result of gene amplification of DHFR gene. In this case, an increase in the expression of the gene product contributes to the acquisition of drug resistance, which suggests a probable connection between transcription and recombination. The effect of MTX on the growth of tsTM4 cells was examined by comparison with that of parental CHO-K1 cells (Fig. S1). Interestingly, the growth of tsTM4 cells appeared to be faster than that of CHO-K1 cells. However, in regard to the mechanism underlying the difference in growth rates, it remains unclear whether the drug resistance was obtained by gene amplification. The difference in the sensitivity to MTX between CHO-K1 and tsTM4 cells will need to be assessed carefully. Amplification of oncogenes such as c-myc is frequently found in many human cancers and is closely associated with the formation of cancer malignancy, which occurs through overproduction of specific protein products (for a review, see [24]).

Splicing

Temperature-sensitive mutant cell tsTM18

tsTM18 is another ts mutant from the CHO-K1 cell line with Smu1 being the gene responsible for this defect (Table 1) [9,11]. The involvement of smu-1 in splicing was revealed by genetic studies of smu-1 in Caenorhabditis elegans [25]. tsTM18 mutant cells at the nonpermissive temperature show an accumulation of single-stranded DNA (ssDNA) in the nucleus, an abnormal chromosome configuration in which the spindle is not completely assembled, and an arrested cell cycle in the S and G2 phases with reduced DNA synthesis and chromosome instability (Table 1) [9,11].

Smu1 and SRSF1

The introduction and expression of a hybrid gene encoding wild-type Smu1 tagged with green fluorescent protein (GFP) can rescue tsTM18 cells to allow growth at 39°C [26]. Analysis of live cells revealed speckle-like concentrations of GFP-Smu1/Smu1-GFP located in discrete nucleoplasmic sites against a diffuse background [26]. These ‘splicing speckles’ are major nuclear domains that contain many components of the splicing machinery [27]. Indirect immunostaining revealed that GFP-tagged Smu1 co-localized in the speckles with SRSF1 (SF2/ASF) [26]. SRSF1, which belongs to the SR splicing group of factors, is a marker of speckles [28,29]. We also found that localization of SRSF1 in the tsTM18 cells cultured at 39°C was altered by the Smu1 ts defect and was dependent on the incubation time at 39°C [26], which again suggests a functional association between Smu1 and SRSF1 [26]. The above results suggested that Smu1 may function to properly localize SRSF1 at the mRNA processing sites.

Chromosome instability caused by the loss of SRSF1

Li and Manley reported a connection between splicing, transcription, and maintenance of genomic stability with regard to the loss of SRSF1 [30]. They showed that SRSF1 had a significant function in maintaining genome stability, that of preventing the formation of R-loop structures (RNA:DNA hybrid) between nascent transcripts and template DNAs. Because the ts defect of Smu1 causes chromosome instability in tsTM18 cells, the association of Smu1 with SRSF1 found in our study proved to be very interesting.

RNAi screening for genome instability

RNAi (RNA interference) has actively allowed the screening of genes whose functions are involved in a particular pathway in cells, such as chromosome instability. RNAi screening revealed that Smu1 is involved in the integrity of both mitotic spindles and genome stability [31,32]. Interestingly, according to these results, the most significantly enriched pathway mediating genome instability includes proteins operating at different stages of mRNA processing, such as RNA splicing and spliceosome assembly [32]. In addition, genes important for transcription and RNA processing appear to be enriched among those genes responding to replication stress [33] (For comprehensive reviews, see [34]).

R-loop and replication stress

When RNA processing components are lost, as with the loss of SRSF1 described above, the nascent transcript may inappropriately re-hybridize with the template DNA, forming a three-stranded nucleic acid structure comprising an RNA:DNA hybrid and a ssDNA that might inhibit replication (see [5,35,36] for comprehensive reviews).

Active pathways exist to prevent replication stress and to resolve the conflicts between transcription and replication (see [35–37] for comprehensive reviews). For example, topoisomerases can help to relieve the topological stress that is generated by replication and transcription complexes [38,39]. RNA processing factors can prevent the nascent transcript from interacting with the template DNA [40,41], and RNase H can also digest the RNA of an RNA:DNA hybrid [30,42]. In addition, it was suggested that histone H3K9 methylation in C. elegans suppresses RNA:DNA hybrid-associated repeat instability [43].

Two novel techniques are available to analyze R-loops. The R-ChIP approach was developed using inactive RNase H to reveal dynamic coupling of R-loops with transcriptional pausing at gene promoters [44]. Similar results were obtained from another technique using an antibody raised against RNA:DNA hybrid, bis DRIP-seq (bisulfite DNA–RNA immunoprecipitation sequencing) [45]. These novel techniques permitting the detection of R-loops in a genome will promote further research into their formation and provide detailed information on the roles played in transcription, chromatin structure, and genomic instability.

Conclusions: perspective on RNA metabolisms and genome

Taken together, our series of studies proved the connection between the defects of RNA metabolism and the induction of chromosome instability [9–11,17]. Our findings clearly showed that genome instability was caused by the links between RNA metabolism and replication, resulting in genomic recombination. Figure 2 represents a model for genome evolution triggered by the defects of RNA metabolism. Defects of RNA synthesis (transcription) can directly affect DNA synthesis (replication) [9,10,17,18,21], resulting in a tug of war between transcription and replication. It has been suggested that the various ways in which the essential process of transcription can change the underlying DNA template and thereby modify the genetic landscape propose transcription as one source of genome instability [21,46–48]. Defects of RNA editing (splicing) can also directly affect DNA synthesis [9,11,30], including the formation of R-loops. Such defects may cause recombination to rescue crises of replication, which may appear as sister chromatid exchanges and gene amplification [9,22]. The R-loop was proposed to be an important component of the class switch recombination mechanism in B cells [49]. The link between transcription, replication, and recombination has been defined as transcription-associated recombination in eukaryotes [50]. Sister chromatid exchange and gene amplification can contribute to genome evolution by introducing diversity and variability, such as the acquisition of resistance against anti-cancer agents. Therefore, our results support the hypothesis that RNA metabolism may be one of the driving forces of genome modification as a trigger of genome evolution.

Figure 2.

Model for genome evolution triggered by the defects of RNA metabolisms. Defects of RNA synthesis (transcriptions) directly affect DNA synthesis (replication), resulting in a tug of war between transcription and replication. Defects of RNA editing (splicing) also directly affect DNA synthesis, resulting in the formation of an R-loop. They may cause recombination to rescue crises of replication, which appear as sister chromatid exchanges and gene amplification, both of which can contribute to genome evolution by introducing diversities and variabilities.

Acknowledgments

The author acknowledges everyone who contributed to our studies on ts mutants, especially Mrs. Yoshie Ishihara for her technical assistance.

Disclosure statement

No potential conflict of interest was reported by the author.

Supplementary Material

Supplemental data for this article can be accessed here.