Emilien Nicolas

Chromosome Replication and Segregation in Bacteria

In dividing cells, chromosome duplication once per generation must be coordinated with faithful segregation of newly replicated chromosomes and with cell growth and division. Many of the mechanistic details of bacterial replication elongation are well established. However, an understanding of the complexities of how replication initiation is controlled and coordinated with other cellular processes is emerging only slowly. In contrast to eukaryotes, in which replication and segregation are separate in time, the segregation of most newly replicated bacterial genetic loci occurs sequentially soon after replication. We compare the strategies used by chromosomes and plasmids to ensure their accurate duplication and segregation and discuss how these processes are coordinated spatially and temporally with growth and cell division. We also describe what is known about the three conserved families of ATP-binding proteins that contribute to chromosome segregation and discuss their inter-relationships in a range of disparate bacteria.

The Tn3-family of Replicative Transposons.

Transposons of the Tn3 family form a widespread and remarkably homogeneous group of bacterial transposable elements in terms of transposition functions and an extremely versatile system for mediating gene reassortment and genomic plasticity owing to their modular organization. They have made major contributions to antimicrobial drug resistance dissemination or to endowing environmental bacteria with novel catabolic capacities. Here, we discuss the dynamic aspects inherent to the diversity and mosaic structure of Tn3-family transposons and their derivatives. We also provide an overview of current knowledge of the replicative transposition mechanism of the family, emphasizing most recent work aimed at understanding this mechanism at the biochemical level. Previous and recent data are put in perspective with those obtained for other transposable elements to build up a tentative model linking the activities of the Tn3-family transposase protein with the cellular process of DNA replication, suggesting new lines for further investigation. Finally, we summarize our current view of the DNA site-specific recombination mechanisms responsible for converting replicative transposition intermediates into final products, comparing paradigm systems using a serine recombinase with more recently characterized systems that use a tyrosine recombinase.

Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress

The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucleolar morphology. Remarkably, we find that uL5 (formerly RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar integrity. Together with the 5S rRNA, they form the late-assembling central protuberance on mature 60S subunits, and act as an Hdm2 trap and p53 stabilizer. Other major contributors to p53 homeostasis are also strictly late-assembling large subunit r-proteins essential to nucleolar structure. The identification of the r-proteins that specifically contribute to maintaining nucleolar structure and p53 steady-state level provides insights into fundamental aspects of cell and cancer biology.

Carboxy-silane coated iron oxide nanoparticles: a convenient platform for cellular and small animal imaging

This study reports the synthesis of stabilized ultrasmall iron oxide nanoparticles (USPIO) as bimodal probes for magnetic resonance and optical imaging. These nanosystems are based on small iron oxide cores surrounded by a thin polysiloxane shell exhibiting carboxylic acid functions. Thanks to these functions, hybrid particles were obtained by conjugating a fluorophore to the superparamagnetic contrastophore. Such modification allowed us to directly follow these USPIO in cellulo, which provided interesting information about their internalization pathway and cellular distribution upon mitosis. Finally, the efficiency of these systems as probes for bimodal imaging was emphasized by the observation of their in vivo behavior in mice using magnetic resonance and optical imaging.

MatP regulates the coordinated action of topoisomerase IV and MukBEF in chromosome segregation.

The Escherichia coli SMC complex, MukBEF, forms clusters of molecules that interact with the decatenase topisomerase IV and which are normally associated with the chromosome replication origin region (ori). Here we demonstrate an additional ATP-hydrolysis-dependent association of MukBEF with the replication termination region (ter). Consistent with this, MukBEF interacts with MatP, which binds matS sites in ter. MatP displaces wild-type MukBEF complexes from ter, thereby facilitating their association with ori, and limiting the availability of topoisomerase IV (TopoIV) at ter. Displacement of MukBEF is impaired when MukB ATP hydrolysis is compromised and when MatP is absent, leading to a stable association of ter and MukBEF. Impairing the TopoIV-MukBEF interaction delays sister ter segregation in cells lacking MatP. We propose that the interplay between MukBEF and MatP directs chromosome organization in relation to MukBEF clusters and associated topisomerase IV, thereby ensuring timely chromosome unlinking and segregation.

Separate structural and functional domains of Tn4430 transposase contribute to target immunity.

Like other transposons of the Tn3 family, Tn4430 exhibits target immunity, a process that prevents multiple insertions of the transposon into the same DNA molecule. Immunity is conferred by the terminal inverted repeats of the transposon and is specific to each element of the family, indicating that the transposase TnpA is directly involved in the process. However, the molecular mechanism whereby this protein promotes efficient transposition into permissive targets while preventing transposition into immune targets remains unknown. Here, we demonstrate that both functions of TnpA can be uncoupled from each other by isolating and characterizing mutants that are proficient in transposition (T+) but impaired in immunity (I‐). The identified T+/I‐ mutations are clustered into separate structural and functional domains of TnpA, indicating that different activities of the protein contribute to immunity. Combination of separate mutations had synergistic effects on target immunity but contrasting effects on transposition. One class of mutations was found to stimulate transposition, whereas other mutations appeared to reduce TnpA activity. The data are discussed with respect to alternative models in which TnpA acts as a specific determinant to both establish and respond to immunity.

Target Immunity of the Tn3-Family Transposon Tn4430 Requires Specific Interactions between the Transposase and the Terminal Inverted Repeats of the Transposon

Specificity of the Tn4430 target immunity signal was examined by fusing the transposase TnpA to the LacI repressor of Escherichia coli. The resulting chimeric proteins failed to impose immunity to DNA targets carrying copies of the lacO operator, though they were proficient in lacO binding in vivo and remained responsive to wild-type immunity conferred by the Tn4430 inverted repeat end. Intriguingly, the presence of lacO repeats within the target was found to strongly influence target site selection by Tn4430, but in a LacI-independent manner.

The human box C/D snoRNAs U3 and U8 are required for pre-rRNA processing and tumorigenesis

Small nucleolar RNAs (snoRNAs) are emerging as a novel class of proto-oncogenes and tumor suppressors; their involvement in tumorigenesis remains unclear. The box C/D snoRNAs U3 and U8 are upregulated in breast cancers. Here we characterize the function of human U3 and U8 in ribosome biogenesis, nucleolar structure, and tumorigenesis. We show in breast (MCF-7) and lung (H1944) cancer cells that U3 and U8 are required for pre-rRNA processing reactions leading, respectively, to synthesis of the small and large ribosomal subunits. U3 or U8 depletion triggers a remarkably potent p53-dependent anti-tumor stress response involving the ribosomal proteins uL5 (RPL11) and uL18 (RPL5). Interestingly, the nucleolar structure is more sensitive to perturbations in lung cancer than in breast cancer cells. We reveal in a mouse xenograft model that the tumorigenic potential of cancer cells is reduced in the case of U3 suppression and totally abolished upon U8 depletion. Tumors derived from U3-knockdown cells displayed markedly lower metabolic volume and activity than tumors derived from aggressive control cancer cells. Unexpectedly, metabolic tracer uptake by U3-suppressed tumors appeared more heterogeneous, indicating distinctive tumor growth properties that may reflect non-conventional regulatory functions of U3 (or fragments derived from it) in mRNA metabolism.

Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly.

Significance The Tn3 family of transposons, discovered in the early 1970s, represents a serious threat to human health because of its prevalence in the dissemination of antibiotic resistance and, indirectly, because of its involvement in xenobiotic metabolism and in the transmission of plant pathogenicity determinants. Astonishingly, their transposition mechanism has yet to be elucidated. We have started to unravel this mechanism by reconstituting the transposition reaction of the Tn3-family transposon Tn4430 in a cell-free in vitro system. The assays also were used to characterize transposase mutants affected in target immunity, a phenomenon whereby a given transposon avoids inserting more than once into the same DNA target. The data support a tentative model linking target immunity with transposition complex assembly and activation. The Tn3 family is a widespread group of replicative transposons that are notorious for their contribution to the dissemination of antibiotic resistance and the emergence of multiresistant pathogens worldwide. The TnpA transposase of these elements catalyzes DNA breakage and rejoining reactions required for transposition. It also is responsible for target immunity, a phenomenon that prevents multiple insertions of the transposon into the same genomic region. However, the molecular mechanisms whereby TnpA acts in both processes remain unknown. Here, we have developed sensitive biochemical assays for the TnpA transposase of the Tn3-family transposon Tn4430 and used these assays to characterize previously isolated TnpA mutants that are selectively affected in immunity. Compared with wild-type TnpA, these mutants exhibit deregulated activities. They spontaneously assemble a unique asymmetric synaptic complex in which one TnpA molecule simultaneously binds two transposon ends. In this complex, TnpA is in an activated state competent for DNA cleavage and strand transfer. Wild-type TnpA can form this complex only on precleaved ends mimicking the initial step of transposition. The data suggest that transposition is controlled at an early stage of transpososome assembly, before DNA cleavage, and that mutations affecting immunity have unlocked TnpA by stabilizing the protein in a monomeric activated synaptic configuration. We propose an asymmetric pathway for coupling active transpososome assembly with proper target recruitment and discuss this model with respect to possible immunity mechanisms.

SAMMSON fosters cancer cell fitness by concertedly enhancing mitochondrial and cytosolic translation

Synchronization of mitochondrial and cytoplasmic translation rates is critical for the maintenance of cellular fitness, with cancer cells being especially vulnerable to translational uncoupling. Although alterations of cytosolic protein synthesis are common in human cancer, compensating mechanisms in mitochondrial translation remain elusive. Here we show that the malignant long non-coding RNA (lncRNA) SAMMSON promotes a balanced increase in ribosomal RNA (rRNA) maturation and protein synthesis in the cytosol and mitochondria by modulating the localization of CARF, an RNA-binding protein that sequesters the exo-ribonuclease XRN2 in the nucleoplasm, which under normal circumstances limits nucleolar rRNA maturation. SAMMSON interferes with XRN2 binding to CARF in the nucleus by favoring the formation of an aberrant cytoplasmic RNA–protein complex containing CARF and p32, a mitochondrial protein required for the processing of the mitochondrial rRNAs. These data highlight how a single oncogenic lncRNA can simultaneously modulate RNA–protein complex formation in two distinct cellular compartments to promote cell growth.The lncRNA SAMMSON promotes a balanced increase in rRNA maturation and protein synthesis in the cytosol and mitochondria by modulating the localization of CARF, which affects two rRNA-processing factors.