Hélène Tourrière

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.

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.

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.

RasGAP-Associated Endoribonuclease G3BP: Selective RNA Degradation and Phosphorylation-Dependent Localization

ABSTRACT Mitogen activation of mRNA decay pathways likely involves specific endoribonucleases, such as G3BP, a phosphorylation-dependent endoribonuclease that associates with RasGAP in dividing but not quiescent cells. G3BP exclusively cleaves between cytosine and adenine (CA) after a specific interaction with RNA through the carboxyl-terminal RRM-type RNA binding motif. Accordingly, G3BP is tightly associated with a subset of poly(A)+ mRNAs containing its high-affinity binding sequence, such as the c-myc mRNA in mouse embryonic fibroblasts. Interestingly, c-myc mRNA decay is delayed in RasGAP-deficient fibroblasts, which contain a defective isoform of G3BP that is not phosphorylated at serine 149. A G3BP mutant in which this serine is changed to alanine remains exclusively cytoplasmic, whereas a glutamate for serine substitution that mimics the charge of a phosphorylated serine is translocated to the nucleus. Thus, a growth factor-induced change in mRNA decay may be modulated by the nuclear localization of a site-specific endoribonuclease such as G3BP.

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.

Control of Fetal Growth and Neonatal Survival by the RasGAP-Associated Endoribonuclease G3BP

ABSTRACT 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.

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.

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.