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The notions of positional information and positional value describe the role of cell position in cell development and pattern formation. Despite their frequent usage in literature, their definitions are blurry, and are interpreted differently by different researchers. Through reflection on previous definitions and usage, and analysis of related experiments, we propose three clear and verifiable criteria for positional information/value. Then we reviewed literature on molecular mechanisms of cell development and pattern formation, to search for a possible molecular basis of positional information/value, including those used in theoretical models. We conclude that although morphogen gradients and cell-to-cell contacts are involved in the pattern formation process, complete molecular explanations of positional information/value are still far from reality.

We review studies on tissue transplantation experiments for various species: one piece of the donor tissue is excised and transplanted into a slit in the host tissue, then observe the behavior of this grafted tissue. Although we have known the results of some transplantation experiments, there are many more possible experiments with unknown results. We develop a penalty function-based method that uses the known experimental results to infer the unknown experimental results. Similar experiments without similar results get penalized and correspond to smaller probability. This method can provide the most probable results of a group of experiments or the probability of a specific result for each experiment. This method is also generalized to other situations. Besides, we solve a problem: how to design experiments so that such a method can be applied most efficiently.

International audience; IGF-2 mRNA binding protein 3 (IGF2BP3, IMP-3) is a well-known post-transcriptional regulatory factor of gene expression, mainly involved in embryonic development and oncogenesis. We have previously demonstrated that a subset of IMP-3 targets, such as the mRNAs of cyclins D1, D3 and G1, are positively regulated by IMP-3, and that this regulation depends on nuclear localization of IMP-3. In the present study, we show that as a first step following a knock-down of IMP-3, the protein levels of the cyclins rapidly decrease, while their mRNAs remain stable and associated with the polyribosomes, though not translated. We have elucidated the molecular mechanisms of this regulation, demonstrating that IMP-3 and its protein partners ILF3/NF90 and PTBP1 bind to the 3'UTRs of the cyclin mRNAs and protect them from the translational repression induced by miRNA-dependent recruitment of AGO2/GW182 complex in human cancer cells.

Functional genomics employs several experimental techniques to investigate gene functions. These techniques such as loss-of-function screening and transcriptome profiling performed in a high-throughput manner give as result a list of genes involved in the biological process of interest. There exist several computational methods for analysis and interpretation of the list. The most widespread methods aim at investigation of biological processes significantly represented in the list or at extracting significantly represented subnetworks. Here we present a new exploratory network analysis method that employs the shortest path approach and centrality measure to uncover members of active molecular pathways leading to the studied phenotype based on the results of functional genomics screening data. We present the method and we demonstrate what data can be retrieved by its application to the terminal muscle differentiation miRNA loss-of-function screening and transcriptomic profiling data and to the ‘druggable’ loss-of-function RNAi screening data of the DNA repair process.

International audience; A genome-wide screen had previously shown that knocking down miR-98 and let-7g, two miRNAs of the let-7 family, leads to a dramatic increase in terminal myogenic differentiation. In the present paper, we report that a transcriptomic analysis of human myoblasts, where miR-98 was knocked down, revealed that approximately 240 genes were sensitive to miR-98 depletion. Among these potential targets of miR-98, we identified the transcriptional repressor E2F5 and showed that it is a direct target of miR-98. Knocking down simultaneously E2F5 and miR-98 almost fully restored normal differentiation, indicating that E2F5 is involved in the regulation of skeletal muscle differentiation. We subsequently show that E2F5 can bind to the promoters of two inhibitors of terminal muscle differentiation, ID1 (inhibitor of DNA binding 1) and HMOX1 (heme oxygenase 1), which decreases their expression in skeletal myoblasts. We conclude that miR-98 regulates muscle differentiation by altering the expression of the transcription factor E2F5 and, in turn, of multiple E2F5 targets.

MiRNAs impact on the control of cell fate by regulating gene expression at the post-transcriptional level. Here, using mammalian muscle differentiation as a model and a phenotypic loss-of-function screen, we explored the function of miRNAs at the genome-wide level. We found that the depletion of a high number of miRNAs (63) impacted on differentiation of human muscle precursors, underscoring the importance of this post-transcriptional mechanism of gene regulation. Interestingly, a comparison with miRNA expression profiles revealed that most of the hit miRNAs did not show any significant variations of expression during differentiation. These constitutively expressed miRNAs might be required for basic and/or essential cell function, or else might be regulated at the post-transcriptional level. MiRNA inhibition yielded a variety of phenotypes, reflecting the widespread miRNA involvement in differentiation. Using a functional screen (the STarS - Suppressor Target Screen – approach, i. e. concomitant knockdown of miRNAs and of candidate target proteins), we discovered miRNA protein targets that are previously uncharacterized controllers of muscle-cell terminal differentiation. Our results provide a strategy for functional annotation of the human miRnome.

Until recently, RNA was considered to be merely a downstream effector of the “noble” genome, the latter having all the information and therefore occupying a position at the very heart of gene regulation, according to the “central dogma” of DNA transcribed into RNA translated into protein. Although we all knew that RNAs also have accessory functions, and that non-coding RNAs intervene at all stages of gene expression, these essential functions were considered to be mere “housekeepers,” and RNA was denied a regulatory role. This dogma was, however, “blown up” several years ago by the concomitant discovery of RNA interference and microRNAs in a model organism, the worm Caenorhabditis elegans. We now know that small regulatory RNAs are widely conserved in plants and animals, and that microRNAs and short interfering RNAs are not the only kinds of regulatory small RNAs that exist. Indeed, the variety of functions in which small non-coding RNAs have been shown to play essential roles has grown rapidly. Basically, they are involved in controlling large genetic programs or large regions of cell genomes, and they participate in determining what is called cell fate, or the balance between cell proliferation, differentiation and death. This equilibrium is strictly controlled under normal conditions, and its deregulation leads to oncogenesis. One of our main interests is the function of microRNAs in mammalian skeletal muscle. We describe here the high-throughput screening strategy used in our laboratory to identify and validate the microRNAs and their specific targets which are essential for muscle cell differentiation.

Post-transcriptional regulation of gene expression by RNA-binding proteins and by small non-coding RNAs plays an important role in cell biology. Our previous results show that in murine skeletal myoblasts, the expression of Pinch-2, a focal adhesion remodeling factor that regulates cell motility, is repressed by an RNA-binding protein IMP-2/Igf2bp2. We now show that the expression of Pinch-2 is also regulated by the miRNA let-7g. Let-7g and IMP-2 repress Pinch-2 expression independently of each other. A knock-down of let-7g leads to an increase in Pinch-2 expression, and to a decrease of cell motility, which can be reversed by a simultaneous knock-down of Pinch-2. We conclude that let-7g controls the motility of mouse myoblasts in cell culture by post-transcriptionally regulating the expression of Pinch-2.