Visualization of oxidized guanine nucleotides accumulation in living cells with split MutT.

Cancer cells produce vast quantities of reactive oxygen species, leading to the accumulation of toxic nucleotides as 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP). The human MTH1 protein catalyzes the hydrolysis of 8-oxo-dGTP, and cancer cells are dependent on MTH1 for their survival. MTH1 inhibitors are possible candidates for a class of anticancer drugs; however, a reliable screening system using live cells has not been developed. Here we report a visualization method for 8-oxo-dGTP and its related nucleotides in living cells. Escherichia coli MutT, a functional homologue of MTH1, is divided into the N-terminal (1-95) and C-terminal (96-129) parts (Mu95 and 96tT, respectively). Mu95 and 96tT were fused to Ash (assembly helper tag) and hAG (Azami Green), respectively, to visualize the nucleotides as fluorescent foci formed upon the Ash-hAG association. The foci were highly increased when human cells expressing Ash-Mu95 and hAG-96tT were treated with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-dGTP. The foci formation by 8-oxo-dG(TP) was strikingly enhanced by the MTH1 knockdown. Moreover, known MTH1 inhibitors and oxidizing reagents also increased foci. This is the first system that visualizes damaged nucleotides in living cells, provides an excellent detection method for the oxidized nucleotides and oxidative stress, and enables high throughput screening for MTH1 inhibitors.

Methylmercury Decreases AMPA Receptor Subunit GluA2 Levels in Cultured Rat Cortical Neurons.

Methylmercury (MeHg) is a well-known environmental pollutant that has harmful effects on the central nervous systems of humans and animals. The molecular mechanisms of MeHg-induced neurotoxicity at low concentrations are not fully understood. Here, we investigated the effects of low-concentration MeHg on the cell viability, Ca2+ homeostasis, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2 levels, which determine Ca2+ permeability of AMPA receptors, in rat primary cortical neurons. Exposure of cortical neurons to 100 and 300 nM MeHg for 7 d resulted in a decrease in GluA2 levels, an increase in basal intracellular Ca2+ concentration, increased phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2 and p38, and decreased cell viability. Moreover, glutamate stimulation exacerbated the decrease in cell viability and increased intracellular Ca2+ levels in MeHg-treated neurons compared to control neurons. MeHg-induced neuronal cell death was ameliorated by 1-naphthyl acetyl spermine, a specific antagonist of Ca2+-permeable, GluA2-lacking AMPA receptors. Our findings raise the possibility that decreased neuronal GluA2 levels and the subsequent increase in intracellular Ca2+ concentration may contribute to MeHg-induced neurotoxicity.

APC/CCdh1 is required for the termination of chromosomal passenger complex activity upon mitotic exit.

During mitosis, the chromosomal passenger complex (CPC) ensures the faithful transmission of the genome. The CPC is composed of the enzymatic component Aurora B (AURKB) and the three regulatory and targeting components borealin, INCENP, and survivin (also known as BIRC5). Although the CPC is known to be involved in diverse mitotic events, it is still unclear how CPC function terminates after mitosis. Here we show that borealin is ubiquitylated by the anaphase promoting complex/cyclosome (APC/C) and its cofactor Cdh1 (also known as FZR1) and is subsequently degraded in G1 phase. Cdh1 binds to regions within the N terminus of borealin that act as a non-canonical degron. Aurora B has also been shown previously to be degraded by the APC/CCdh1 from late mitosis to G1. Indeed, Cdh1 depletion sustains an Aurora B activity with stable levels of borealin and Aurora B throughout the cell cycle, and causes reduced efficiency of DNA replication after release from serum starvation. Notably, inhibition of Aurora B kinase activity improves the efficiency of DNA replication in Cdh1-depleted cells. We thus propose that APC/CCdh1 terminates CPC activity upon mitotic exit and thereby contributes to proper control of DNA replication.

Hypertension and diabetes mellitus are associated with high FIB-4 index in a health checkup examination cohort without known liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is usually asymptomatic and lacks a specific biomarker; therefore, many individuals might remain undiagnosed even with advanced liver fibrosis. The aim of this study was to clarify the prevalence and clinical features of subjects with a high risk of advanced liver fibrosis in the general population, using the Fibrosis-4 (FIB-4) index. METHODS: We retrospectively investigated 6,087 subjects without known liver disease who had participated in an annual health checkup examination. We analyzed the factors associated with high FIB-4 index (≥ 2.67) using a logistic regression analysis. RESULTS: Among the 6,087 subjects, 76 (1.2%) had high FIB-4 index. Multivariate analysis identified hypertension (odds ratio [OR]; 9.040; 95% confidence interval [CI], 4.081-20.024; P < 0.001) and diabetes mellitus (OR = 4.251; 95% CI, 1.773-10.193; P = 0.001) as important risk factors for high FIB-4 index. The rates of hypertension and diabetes mellitus in subjects with high FIB-4 index were 78.9% and 23.7%, respectively. No significant association was observed between obesity or large waist circumference and high FIB-4 index. A history of cardiovascular disease was significantly more common in subjects with high FIB-4 index. These results were also observed in subjects with normal liver function test. CONCLUSIONS: The present study revealed that approximately 1% of the general Japanese population has a high risk of advanced liver fibrosis. Many of these patients had hypertension and/or diabetes mellitus. Our findings suggest that there are many undiagnosed patients NAFLD with risk of advanced liver fibrosis in the general population.

Preferential digestion of PCNA-ubiquitin and p53-ubiquitin linkages by USP7 to remove polyubiquitin chains from substrates.

Ubiquitin-specific protease 7 (USP7) regulates various cellular pathways through its deubiquitination activity. Despite the identification of a growing number of substrates of USP7, the molecular mechanism by which USP7 removes ubiquitin chains from polyubiquitinated substrates remains unexplored. The present study investigated the mechanism underlying the deubiquitination of Lys63-linked polyubiquitinated proliferating cell nuclear antigen (PCNA). Biochemical analyses demonstrated that USP7 efficiently removes polyubiquitin chains from polyubiquitinated PCNA by preferential cleavage of the PCNA-ubiquitin linkage. This property was largely attributed to the poor activity toward Lys63-linked ubiquitin chains. The preferential cleavage of substrate-ubiquitin linkages was also observed for Lys48-linked polyubiquitinated p53 because of the inefficient cleavage of the Lys48-linked ubiquitin chains. The present findings suggest a mechanism underlying the removal of polyubiquitin signals by USP7.

Stepwise multipolyubiquitination of p53 by the E6AP-E6 ubiquitin ligase complex.

The human papillomavirus (HPV) oncoprotein E6 specifically binds to E6AP (E6-associated protein), a HECT (homologous to the E6AP C terminus)-type ubiquitin ligase, and directs its ligase activity toward the tumor suppressor p53. To examine the biochemical reaction in vitro, we established an efficient reconstitution system for the polyubiquitination of p53 by the E6AP-E6 complex. We demonstrate that E6AP-E6 formed a stable ternary complex with p53, which underwent extensive polyubiquitination when the isolated ternary complex was incubated with E1, E2, and ubiquitin. Mass spectrometry and biochemical analysis of the reaction products identified lysine residues as p53 ubiquitination sites. A p53 mutant with arginine substitutions of its 18 lysine residues was not ubiquitinated. Analysis of additional p53 mutants retaining only one or two intact ubiquitination sites revealed that chain elongation at each of these sites was limited to 5-6-mers. We also determined the size distribution of ubiquitin chains released by en bloc cleavage from polyubiquitinated p53 to be 2-6-mers. Taken together, these results strongly suggest that p53 is multipolyubiquitinated with short chains by E6AP-E6. In addition, analysis of growing chains provided strong evidence for step-by-step chain elongation. Thus, we hypothesize that p53 is polyubiquitinated in a stepwise manner through the back-and-forth movement of the C-lobe, and the permissive distance for the movement of the C-lobe restricts the length of the chains in the E6AP-E6-p53 ternary complex. Finally, we show that multipolyubiquitination at different sites provides a signal for proteasomal degradation.

Spironolactone-induced XPB degradation depends on CDK7 kinase and SCFFBXL18 E3 ligase.

The multisubunit complex transcription factor IIH (TFIIH) has dual functions in transcriptional initiation and nucleotide excision repair (NER). TFIIH is comprised of two subcomplexes, the core subcomplex (seven subunits) including XPB and XPD helicases and the cyclin-dependent kinase (CDK)-activating kinase (CAK) subcomplex (three subunits) containing CDK7 kinase. Recently, it has been reported that spironolactone, an anti-aldosterone drug, inhibits cellular NER by inducing proteasomal degradation of XPB and potentiates the cytotoxicity of platinum-based drugs in cancer cells, suggesting possible drug repositioning. In this study, we have tried to uncover the mechanism underlying the chemical-induced XPB destabilization. Based on siRNA library screening and subsequent analyses, we identified SCFFBXL18 E3 ligase consisting of Skp1, Cul1, F-box protein FBXL18 and Rbx1 responsible for spironolactone-induced XPB polyubiquitination and degradation. In addition, we showed that CDK7 kinase activity is required for this process. Finally, we found that the Ser90 residue of XPB is essential for the chemical-induced destabilization. These results led us to propose a model that spironolactone may trigger the phosphorylation of XPB at Ser90 by CDK7, which promotes the recognition and polyubiquitination of XPB by SCFFBXL18 for proteasomal degradation.

Replication stress induces accumulation of FANCD2 at central region of large fragile genes.

During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.

Overexpression of Rev1 promotes the development of carcinogen-induced intestinal adenomas via accumulation of point mutation and suppression of apoptosis proportionally to the Rev1 expression level.

Cancer development often involves mutagenic replication of damaged DNA by the error-prone translesion synthesis (TLS) pathway. Aberrant activation of this pathway plays a role in tumorigenesis by promoting genetic mutations. Rev1 controls the function of the TLS pathway, and Rev1 expression levels are associated with DNA damage induced cytotoxicity and mutagenicity. However, it remains unclear whether deregulated Rev1 expression triggers or promotes tumorigenesis in vivo. In this study, we generated a novel Rev1-overexpressing transgenic (Tg) mouse and characterized its susceptibility to tumorigenesis. Using a small intestinal tumor model induced by N-methyl-N-nitrosourea (MNU), we found that transgenic expression of Rev1 accelerated intestinal adenoma development in proportion to the Rev1 expression level; however, overexpression of Rev1 alone did not cause spontaneous development of intestinal adenomas. In Rev1 Tg mice, MNU-induced mutagenesis was elevated, whereas apoptosis was suppressed. The effects of hREV1 expression levels on the cytotoxicity and mutagenicity of MNU were confirmed in the human cancer cell line HT1080. These data indicate that dysregulation of cellular Rev1 levels leads to the accumulation of mutations and suppression of cell death, which accelerates the tumorigenic activities of DNA-damaging agents.

Selective enhancing effect of early mitotic inhibitor 1 (Emi1) depletion on the sensitivity of doxorubicin or X-ray treatment in human cancer cells.

Chemotherapy and radiation in addition to surgery has proven useful in a number of different cancer types, but the effectiveness in normal tissue cannot be avoided in these therapies. To improve the effectiveness of these therapies selectively in cancer tissue is important for avoiding side effects. Early mitotic inhibitor 1 (Emi1) is known to have the function to inhibit anaphase-promoting complex/cyclosome ubiquitin ligase complex, which ubiquitylates the cell cycle-related proteins. It recently has been shown that Emi1 knockdown prevents transition from S to G2 phase by down-regulating geminin via anaphase-promoting complex/cyclosome activation. At present, anticancer drugs for targeting DNA synthesis to interfere with rapidly dividing cells commonly are used. As Emi1 depletion interferes with completion of DNA synthesis in cancer cells, we thought that Emi1 knockdown might enhance the sensitivity for anticancer agents. Here, we confirmed that Emi1 siRNA induced polyploidy for preventing transition from S to G2 phase in several cancer cell lines. Then, we treated Emi1 depleted cells with doxorubicin. Interestingly, increased apoptotic cells were observed after doxorubicin treatment in Emi1 siRNA-treated cancer cells. In addition, Emi1 depletion enhanced the sensitivity of x-ray irradiation in cancer cells. Importantly, synergistic effect of Emi1 knockdown in these combination therapies was not observed in normal cells. These results suggest that Emi1 siRNA can be a useful tool for enhancing of sensitivity of cancer cells to anticancer reagents and radiation.

Asymmetric nature of two subunits of RAD18, a RING-type ubiquitin ligase E3, in the human RAD6A-RAD18 ternary complex.

RAD18, a RING-type ubiquitin ligase (E3) that plays an essential role in post-replication repair, possesses distinct domains named RING, UBZ, SAP and the RAD6-binding domain (R6BD) and forms a dimer. RAD6, an ubiquitin-conjugating enzyme (E2), stably associates with R6BD in the C-terminal portion. In this study, we established a method to distinguish between the two subunits of RAD18 by introduction of different tags, and analyzed mutant complexes. Our results, surprisingly, demonstrate that RAD6A and RAD18 form a ternary complex, RAD6A-(RAD18)(2) and the presence of only one R6BD in the two RAD18 subunits is sufficient for ternary complex formation and the ligase activity. Interestingly, ligase activity of a mutant dimer lacking both R6BDs is not restored even with large amounts of RAD6A added in solution, suggesting a requirement for precise juxtaposition via interaction with R6BD. We further show that mutations in both subunits of either RING or SAP, but not UBZ, strongly reduce ligase activity, although inactivation in only one of two subunits is without effect. These results suggest an asymmetric nature of the two RAD18 subunits in the complex.

En bloc transfer of polyubiquitin chains to PCNA in vitro is mediated by two different human E2-E3 pairs.

Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6-RAD18 (an E2-E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2-UBC13 (a UEV-E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13∼Ubn) and then transfers the chain to RAD6∼Ub, forming RAD6∼Ubn+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication.

The p53 inhibitors MDM2/MDMX complex is required for control of p53 activity in vivo.

There are currently two distinct models proposed to explain why both MDM2 and MDMX are required in p53 control, with a key difference centered on whether these two p53 inhibitors work together or independently. To test these two competing models, we generated knockin mice expressing a point mutation MDMX mutant (C462A) that is defective in MDM2 binding. This approach allowed a targeted disassociation of the MDM2/MDMX heterocomplex without affecting the ability of MDMX to bind to p53, and while leaving the MDM2 protein itself completely untouched. Significantly, Mdmx(C462A/C462A) homozygous mice died at approximately day 9.5 of embryonic development, as the result of a combination of apoptosis and decreased cell proliferation, as shown by TUNEL and BrdU incorporation assays, respectively. Interestingly, even though the MDMX mutant protein abundance was found slightly elevated in the Mdmx(C462A/C462A) homozygous embryos, both the abundance and activity of p53 were markedly increased. A p53-dependent death was demonstrated by the finding that concomitant deletion of p53 completely rescued the embryonic lethality in Mdmx(C462A/C462A) homozygous mice. Our data demonstrate that MDM2 and MDMX function as an integral complex in p53 control, providing insights into the nonredundant nature of the function of MDM2 and MDMX.

Correction of substitution, deletion, and insertion mutations by 5'-tailed duplexes.

Germline and somatic mutations cause various diseases, including cancer. Clinical applications of genome editing are keenly anticipated, since it can cure genetic diseases. Recently, we reported that a 5'-tailed duplex (TD), consisting of an approximately 80-base editor strand oligodeoxyribonucleotide and a 35-base assistant strand oligodeoxyribonucleotide, could edit a target gene on plasmid DNA and correct a single-base substitution mutation without an artificial nuclease in human cells. In this study, we assessed the ability of the TD to correct base substitution mutations located consecutively or separately, and deletion and insertion mutations. A TD with an 80-base editor strand was co-introduced into human U2OS cells with plasmid DNA bearing either a wild-type or mutated copepod green fluorescent protein (copGFP) gene. Among the mutations, three-base consecutive substitutions were efficiently repaired. The correction efficiencies of deletion mutations were similar to those of substitution mutations, and two to three times higher than those of insertion mutations. Up to three-base substitution, deletion, and insertion mutations were excellent targets for correction by TDs. These results suggested that the TDs are useful for editing disease-causing genes with small mutations.

Phosphorylation by aurora kinase A induces Mdm2-mediated destabilization and inhibition of p53.

Aurora kinase A (also called STK15 and BTAK) is overexpressed in many human cancers. Ectopic overexpression of aurora kinase A in mammalian cells induces centrosome amplification, chromosome instability and oncogenic transformation, a phenotype characteristic of loss-of-function mutations of p53. Here we show that aurora kinase A phosphorylates p53 at Ser315, leading to its ubiquitination by Mdm2 and proteolysis. p53 is not degraded in the presence of inactive aurora kinase A or ubiquitination-defective Mdm2. Destabilization of p53 by aurora kinase A is abrogated in the presence of mutant Mdm2 that is unable to bind p53 and after repression of Mdm2 by RNA interference. Silencing of aurora kinase A results in less phosphorylation of p53 at Ser315, greater stability of p53 and cell-cycle arrest at G2-M. Cells depleted of aurora kinase A are more sensitive to cisplatin-induced apoptosis, and elevated expression of aurora kinase A abolishes this response. In a sample of bladder tumors with wild-type p53, elevated expression of aurora kinase A was correlated with low p53 concentration. We conclude that aurora kinase A is a key regulatory component of the p53 pathway and that overexpression of aurora kinase A leads to increased degradation of p53, causing downregulation of checkpoint-response pathways and facilitating oncogenic transformation of cells.

Correction of monomeric enhanced green fluorescent protein (mEGFP) gene by short 5'-tailed duplexes.

Mutations of important genes elicit various disorders, including cancer. Recently, a new version of a 5'-tailed duplex (short TD), consisting of a ∼100-base editor strand containing the wild-type sequence and a ∼35-base assistant strand, was shown to correct a base substitution mutation in a target gene in human cells. In that previous study, the target was the copepod green fluorescent protein (copGFP) gene. To examine the usefulness of the short TD, we performed gene correction experiments using a mutant form of the monomeric enhanced Aequorea victoria green fluorescent protein (mEGFP) gene containing a TAC to CAC mutation in codon 75 (corresponding to the tyrosine to histidine substitution in the chromophore). The short TDs with the wild-type sequence efficiently corrected the inactivated gene in human U2OS cells. These results indicated that the short TDs are effective for gene editing.

RFWD3 and translesion DNA polymerases contribute to PCNA modification-dependent DNA damage tolerance.

DNA damage tolerance pathways are regulated by proliferating cell nuclear antigen (PCNA) modifications at lysine 164. Translesion DNA synthesis by DNA polymerase η (Polη) is well studied, but less is known about Polη-independent mechanisms. Illudin S and its derivatives induce alkyl DNA adducts, which are repaired by transcription-coupled nucleotide excision repair (TC-NER). We demonstrate that in addition to TC-NER, PCNA modification at K164 plays an essential role in cellular resistance to these compounds by overcoming replication blockages, with no requirement for Polη. Polκ and RING finger and WD repeat domain 3 (RFWD3) contribute to tolerance, and are both dependent on PCNA modifications. Although RFWD3 is a FANC protein, we demonstrate that it plays a role in DNA damage tolerance independent of the FANC pathway. Finally, we demonstrate that RFWD3-mediated cellular survival after UV irradiation is dependent on PCNA modifications but is independent of Polη. Thus, RFWD3 contributes to PCNA modification-dependent DNA damage tolerance in addition to translesion DNA polymerases.

Low-Dose-Rate Irradiation Suppresses the Expression of Cell Cycle-Related Genes, Resulting in Modification of Sensitivity to Anti-Cancer Drugs.

The biological effects of low-dose-rate (LDR) radiation exposure in nuclear power plant accidents and medical uses of ionizing radiation (IR), although being a social concern, remain unclear. In this study, we evaluated the effects of LDR-IR on global gene expression in human cells and aimed to clarify the mechanisms. RNA-seq analyses demonstrated that relatively low dose rates of IR modify gene expression levels in TIG-3 cells under normoxic conditions, but those effects were attenuated under hypoxia-mimicking conditions. Gene set enrichment analysis demonstrated that LDR-IR significantly decreased gene expression related to cell division, cell cycle, mitosis, and the Aurora kinase B and FOXM1 pathways. Quantitative RT-PCR confirmed the down-regulation of AURKB and FOXM1 genes in TIG-3 cells with LDR-IR or hypoxia-mimicking treatments without any dose-rate effect. Knock-down experiments suggested that HIF-1α and HIF-2α, as well as DEC1, participated in down-regulation of AURKB and FOXM1 under DFOM treatments, but to a lesser extent under LDR-IR treatment. FACS and microscopic analyses demonstrated that LDR-IR induced G0/G1 arrest and increased micronucleus or chromosome condensation. Finally, MTT assays demonstrated that LDR-IR decreased sensitivity to paclitaxel or barasertib in TIG-3 cells but not in A549 cells. In conclusion, LDR-IR modifies global gene expression and cell cycle control, resulting in a reduction of sensitivity to anti-cancer chemotherapy in non-cancer cells and thus a reduction in untoward effects (GA).

Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli.

A forward mutagenesis assay using the supF gene has been widely employed for the last several decades in studies addressing mutation frequencies and mutation spectra associated with various intrinsic and environmental mutagens. In this study, by using a supF shuttle vector and non-SOS-induced Escherichia coli with short-read next-generation sequencing (NGS) technology, we present an advanced method for the study of mutations, which is simple, versatile, and cost-effective. We demonstrate the performance of our newly developed assay via pilot experiments with ultraviolet (UV) irradiation, the results from which emerge more relevant than expected. The NGS data obtained from samples of the indicator E. coli grown on titer plates provides mutation frequency and spectrum data, and uncovers obscure mutations that cannot be detected by a conventional supF assay. Furthermore, a very small amount of NGS data from selection plates reveals the almost full spectrum of mutations in each specimen and offers us a novel insight into the mechanisms of mutagenesis, despite them being considered already well known. We believe that the method presented here will contribute to future opportunities for research on mutagenesis, DNA repair, and cancer.

DDX41 coordinates RNA splicing and transcriptional elongation to prevent DNA replication stress in hematopoietic cells.

Myeloid malignancies with DDX41 mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5' splice site (5'SS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5'SS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated with DDX41 mutations.