The RasGAP-associated endoribonuclease G3BP mediates stress granule assembly.

Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149 whose dephosphorylation is induced by arsenite treatment. Critically, a non-phosphorylatable G3BP mutant (S149A) oligomerizes and assembles SG. These results suggest that G3BP is an effector of SG assembly and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.

Reply to "Phosphorylation of G3BP1-S149 does not influence stress granule assembly".

In this issue, Panas et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201801214) challenge the data published in the Tourrière et al. (2013. J. Cell Biol. https://doi.org/10.1083/jcb.200212128) paper on the role of G3BP phosphorylation in stress granule (SG) assembly. This reply addresses that letter and suggests that more work is needed to understand the role of this modification in SG formation.

Overexpression of the Fork Protection Complex: a strategy to tolerate oncogene-induced replication stress in cancer cells.

Oncogene-induced replication stress (RS) plays an active role in tumorigenesis by promoting genomic instability but is also a challenge for cell proliferation. Recent evidence indicates that different types of cancer cells adapt to RS by overexpressing components of the ATR-CHK1 pathway that promote fork progression in a checkpoint-independent manner.

Overexpression of Claspin and Timeless protects cancer cells from replication stress in a checkpoint-independent manner.

Oncogene-induced replication stress (RS) promotes cancer development but also impedes tumor growth by activating anti-cancer barriers. To determine how cancer cells adapt to RS, we have monitored the expression of different components of the ATR-CHK1 pathway in primary tumor samples. We show that unlike upstream components of the pathway, the checkpoint mediators Claspin and Timeless are overexpressed in a coordinated manner. Remarkably, reducing the levels of Claspin and Timeless in HCT116 cells to pretumoral levels impeded fork progression without affecting checkpoint signaling. These data indicate that high level of Claspin and Timeless increase RS tolerance by protecting replication forks in cancer cells. Moreover, we report that primary fibroblasts adapt to oncogene-induced RS by spontaneously overexpressing Claspin and Timeless, independently of ATR signaling. Altogether, these data indicate that enhanced levels of Claspin and Timeless represent a gain of function that protects cancer cells from of oncogene-induced RS in a checkpoint-independent manner.

Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing.

The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart.

Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing.

DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA.

DNA replication is altered in Immunodeficiency Centromeric instability Facial anomalies (ICF) cells carrying DNMT3B mutations.

ICF syndrome is a rare autosomal recessive disorder that is characterized by Immunodeficiency, Centromeric instability, and Facial anomalies. In all, 60% of ICF patients have mutations in the DNMT3B (DNA methyltransferase 3B) gene, encoding a de novo DNA methyltransferase. In ICF cells, constitutive heterochromatin is hypomethylated and decondensed, metaphase chromosomes undergo rearrangements (mainly involving juxtacentromeric regions), and more than 700 genes are aberrantly expressed. This work shows that DNA replication is also altered in ICF cells: (i) heterochromatic genes replicate earlier in the S-phase; (ii) global replication fork speed is higher; and (iii) S-phase is shorter. These replication defects may result from chromatin changes that modify DNA accessibility to the replication machinery and/or from changes in the expression level of genes involved in DNA replication. This work highlights the interest of using ICF cells as a model to investigate how DNA methylation regulates DNA replication in humans.

Exo1 competes with repair synthesis, converts NER intermediates to long ssDNA gaps, and promotes checkpoint activation.

Ultraviolet (UV) light induces DNA-damage checkpoints and mutagenesis, which are involved in cancer protection and tumorigenesis, respectively. How cells identify DNA lesions and convert them to checkpoint-activating structures is a major question. We show that during repair of UV lesions in noncycling cells, Exo1-mediated processing of nucleotide excision repair (NER) intermediates competes with repair DNA synthesis. Impediments of the refilling reaction allow Exo1 to generate extended ssDNA gaps, detectable by electron microscopy, which drive Mec1 kinase activation and will be refilled by long-patch repair synthesis, as shown by DNA combing. We provide evidence that this mechanism may be stimulated by closely opposing UV lesions, represents a strategy to redirect problematic repair intermediates to alternative repair pathways, and may also be extended to physically different DNA damages. Our work has significant implications for understanding the coordination between repair of DNA lesions and checkpoint pathways to preserve genome stability.

Does interference between replication and transcription contribute to genomic instability in cancer cells?

We have recently reported that topoisomerase 1 (Top1) cooperates with ASF/SF2, a splicing factor of the SR family, to prevent unscheduled replication fork arrest and genomic instability in human cells. Our results suggest that Top1 execute this function by suppressing the formation of DNA-RNA hybrids during transcription, these so-called R-loops interfering with the progression of replication forks. Using ChIP-chip, we have shown that γ-H2AX, a marker of DNA damage, accumulates at gene-rich regions of the genome in Top1-deficient cells. This is best illustrated at histone genes, which are highly expressed during S phase and display discrete γ-H2AX peaks on ChIP-chip profiles. Here, we show that these γ-H2AX domains are different from those induced by camptothecin, a Top1 inhibitor inducing double-strand DNA breaks throughout the genome. These data support the view that R-loops promote genomic instability at specific sites by blocking fork progression and inducing chromosome breaks. Whether this type of transcription-dependent fork arrest contributes to the replication stress observed in precancerous lesions is an important question that deserves further attention.

Defining replication origin efficiency using DNA fiber assays.

The timely duplication of eukaryotic genomes depends on the coordinated activation of thousands of replication origins distributed along the chromosomes. Origin activation follows a temporal program that is imposed by the chromosomal context and is under the control of S-phase checkpoints. Although the general mechanisms regulating DNA replication are now well-understood at the level of individual origins, little is known on the coordination of thousands of initiation events at a genome-wide level. Recent studies using DNA combing and other single-molecule assays have shown that eukaryotic genomes contain a large excess of replication origins. Most of these origins remain "dormant" in normal growth conditions but are activated when fork progression is impeded. In this review, we discuss how DNA fiber technologies have changed our view of eukaryotic replication programs and how origin redundancy contributes to the maintenance of genome integrity in eukaryotic cells.

Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription.

Topoisomerase I (Top1) is a key enzyme in functioning at the interface between DNA replication, transcription and mRNA maturation. Here, we show that Top1 suppresses genomic instability in mammalian cells by preventing a conflict between transcription and DNA replication. Using DNA combing and ChIP (chromatin immunoprecipitation)-on-chip, we found that Top1-deficient cells accumulate stalled replication forks and chromosome breaks in S phase, and that breaks occur preferentially at gene-rich regions of the genome. Notably, these phenotypes were suppressed by preventing the formation of RNA-DNA hybrids (R-loops) during transcription. Moreover, these defects could be mimicked by depletion of the splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2), which interacts functionally with Top1. Taken together, these data indicate that Top1 prevents replication fork collapse by suppressing the formation of R-loops in an ASF/SF2-dependent manner. We propose that interference between replication and transcription represents a major source of spontaneous replication stress, which could drive genomic instability during the early stages of tumorigenesis.

Maintenance of fork integrity at damaged DNA and natural pause sites.

S phase is a period of great vulnerability for the genome of eukaryotic cells. Many complicated processes are undertaken during this critical phase of the cell cycle, including the complete unwinding and the duplication of enormously complex DNA molecules. During this process, replication forks frequently encounter obstacles that impede their progression. Arrested forks are unstable structures that have to be stabilized and restarted in order to prevent the formation of double-strand breaks and/or unscheduled homologous recombination. To this aim, cells have evolved complex surveillance mechanisms sensing DNA damage and replication stress. The past decade has seen a dramatic advance in our understanding of how these regulatory pathways act in response to exogenous replication stress. However, the mechanism by which fork integrity is maintained at natural replication-impeding sequences remains obscure. Here, we discuss recent findings about how checkpoint-dependent and -independent mechanisms cooperate to prevent genomic instability at stalled forks, both in normal S phase and in the presence of exogenous genotoxic stress.

Control of fetal growth and neonatal survival by the RasGAP-associated endoribonuclease G3BP.

The regulation of mRNA stability plays a major role in the control of gene expression during cell proliferation, differentiation, and development. Here, we show that inactivation of the RasGAP-associated endoribonuclease (G3BP)-encoding gene leads to embryonic lethality and growth retardation. G3BP-/- mice that survived to term exhibited increased apoptotic cell death in the central nervous system and neonatal lethality. Both in mouse embryonic fibroblasts and during development, the absence of G3BP altered the expression of essential growth factors, among which imprinted gene products and growth arrest-specific mRNAs were outstanding. The results demonstrate that G3BP is essential for proper embryonic growth and development by mediating the coordinate expression of multiple imprinted growth-regulatory transcripts.

Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53.

The yeast checkpoint factors Mrc1p and Tof1p travel with the replication fork and mediate the activation of the Rad53p kinase in response to a replication stress. We show here that both proteins are required for normal fork progression but play different roles at stalled forks. Tof1p is critical for the activity of the rDNA replication fork barrier (RFB) but plays a minor role in the replication checkpoint. In contrast, Mrc1p is not necessary for RFB activity but is essential to mediate the replication stress response. Interestingly, stalled forks did not collapse in mrc1Delta cells exposed to hydroxyurea (HU) as they do in rad53 mutants. However, forks failed to restart when mrc1Delta cells were released from the block. The critical role of Mrc1p in HU is therefore to promote fork recovery in a Rad53p-independent manner, presumably through the formation of a stable fork-pausing complex.

Regulation of cytoplasmic stress granules by apoptosis-inducing factor.

Stress granules (SG) are dynamic cytoplasmic foci in which stalled translation initiation complexes accumulate. In conditions of acute cellular redox, stress cells manipulated to lose the expression of apoptosis-inducing factor (AIF) nucleate SG signature proteins (e.g. TIA-1, PABP1) more efficiently than AIF-positive controls. AIF also inhibited SG formation induced by the RasGAP-associated endoribonuclease G3BP. Retransfection of mouse AIF into cells subjected to human AIF-specific siRNA revealed that only AIF imported into mitochondria could repress SGs and that redox-active domains of AIF, which are dispensable for its apoptogenic action, were required for SG inhibition. In response to oxidative stress, AIF-negative cells were found to deplete non-oxidized glutathione more rapidly than AIF-expressing cells. Exogenous supplementation of glutathione inhibited SG formation elicited by arsenate or G3BP. Together, these data suggest that the oxidoreductase function of AIF is required for the maintenance of glutathione levels in stress conditions and that glutathione is a major regulator of SG.

The RasGAP-associated endoribonuclease G3BP assembles stress granules.

Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents, and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149, whose dephosphorylation is induced by arsenite treatment. Critically, a phosphomimetic mutant (S149E) fails to oligomerize and to assemble SGs, whereas a nonphosphorylatable G3BP mutant (S149A) does both. These results suggest that G3BP is an effector of SG assembly, and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.

mRNA degradation machines in eukaryotic cells.

The steady-state levels of mRNAs depend upon their combined rates of synthesis and processing, transport from the nucleus to cytoplasm, and decay in the cytoplasm. In eukaryotic cells, the degradation of mRNA is an essential determinant in the regulation of gene expression, and it can be modulated in response to developmental, environmental, and metabolic signals. This level of regulation is particularly important for proteins that are active for a brief period, such as growth factors, transcription factors, and proteins that control cell cycle progression. The mechanisms by which mRNAs are degraded and the sequence elements within the mRNAs that affect their stability are the subject of this review. We will summarize the current state of knowledge regarding cis-acting elements in mRNA and trans-acting factors that contribute to mRNA regulation decay. We will then consider the mechanisms by which specific signaling proteins seem to contribute to a dynamic organization of the mRNA degradation machinery in response to physiological stimuli.