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International audience; Notre vie est à l'origine de notre pensée. Elle nous donne notre légitimité à dire le monde parce qu'elle nous y inscrit de fait. Elle est la source de nos interrogations et la matrice de nos catégories de pensée. Dans une sorte de mise en abyme, nous proposons de discuter la place du biographique dans la pratique scientifique. Si l'activité scientifique consiste à tendre vers un « procès sans sujet », les savoirs produits contiennent une dimension subjective, consciente et inconsciente, qui se nourrit de et en même temps sous-tend la biographie du sujet épistémique. Faire de la science suppose une attitude déterminée, un effort continu, un travail. Au fond, on entre en science un peu comme on entre dans les ordres. On cherche la lumière, on avance d'un éclairage – sur le monde et sur soi – vers un autre, et c'est dans la retenue vis-à-vis de la narration de soi, dans la maîtrise de l'expression de ses affects comme de ses désirs, dans la forme que prend dans le discours l'invisibilisation de soi, qu'on se dirige vers la Science. Or, il n'y a pas de savoir scientifique sans sujet épistémique, ni sans sujet tout court, donc sans réflexivité, sans travail sur soi, sans biographie du sujet-cherchant.Dans le cadre du projet scientifique de l'UMR Passages, j'anime depuis 2016 un séminaire sur les présentations poétiques du monde. Il a pour objet le sujet-cherchant et son rapport au monde. Au cours de mes recherches sur la lecture à voix haute, j'ai appris que, pour que les auditeurs trouvent un sens à la lecture, il faut que le texte passe par, au travers du lecteur, par son intériorité, mais sans expression d'émotion. Le lecteur fait un travail, en amont de la lecture, où il ranime des expériences passées, incorporées, sensibles, émouvantes, parfois douloureuses, en rapport avec le texte. Par une mise en images, le lecteur met à distance cette sensibilité première mais nécessaire dans le premier temps et peut alors interpeler son auditoire, lui transmettre du sens à travers un texte qu'il a ainsi fait sien. La proposition que le lecteur fait au public est une présentation poétique du monde, où poétique est à comprendre ici dans une dimension créative, de façonnement, contenant la continuité, la complexité à la fois de son monde et du contexte de la lecture, du rapport lecteur-texte-public-lieu. De façon similaire, le sujet-cherchant livre un discours à son auditoire ou à son lectorat sur un objet choisi et appréhendé à partir d'une formation, de lectures, d'un cadre académique, d'un terrain, et à travers une histoire et un vécu personnel, sensible, tout ceci impliquant toute sa personne, imprégnée d'expériences de vie et de connaissances, c'est-à-dire une personne à la fois singulière, une et ordinaire. C'est dans la forme que prend la résistance de cette unité du sujet-cherchant que réside le poétique, dans la constitution de soi comme de son effacement.Au-delà de leur enjeu épistémologique, ces travaux sont menés dans une perspective éthique. Prendre conscience du poétique en science, en témoigner et en rendre compte contribue à l'humanisation de la science et de ses travailleurs.

International audience; Chez l’homme, les études d’association pangénomiques basées sur les programmes génomiques institutionnels et commerciaux permettent aujourd’hui d’identifier des polymorphismes génétiques (SNPs) associés à des traits complexes de nature psychosociale et déterminés par des centaines de gènes différents (le niveau d’instruction, l’intelligence, des traits psychotiques, névrotiques et émotionnels…). L’enjeu immédiat n’est plus l’identification du lien causal et mécanistique qui explique la contribution d’un SNP à un trait mais la construction d’outils métrologiques nouveaux désignés scores polygéniques pangénomiques. Ces scores proposent d’exprimer en une valeur numérique unique la capacité d’un génome donné à produire un trait complexe. Ces scores deviennent des outils analytiques et prédictifs censés traduire la potentialité génétique d’une personne par rapport à ces traits psychosociaux et conduisent ainsi à une biologisation statistique de « l’exception humaine ».

International audience; Dans le phénomène de la vie, Hans Jonas introduit une ontologie de la vie organique comme l’exercice d’une liberté nécessiteuse centrée sur le métabolisme. Dans un bref appendice du troisième essai qui compose l’ouvrage, Jonas pose que l’ADN et les cellules neuronales seraient exclus du métabolisme et constitueraient ainsi des exceptions comme invariants matériels. Nous analysons ce texte pour tenter de préciser la conception jonassienne de la vie organique. Nous posons ainsi, qu’à contre-courant de cet appendice (mais en accord avec la pensée générale de Jonas), ni l’ADN, ni les neurones ne peuvent être considérés comme des invariants matériels stricts. Nous soulignons aussi la déflation, dans cet appendice, de certains aspects de la vie organique comme la croissance, le développement et surtout l’évolution au profit d’une prise en compte de l’organisme immédiat. Nous proposons qu’avec Jonas une articulation des perspectives biologiques et phénoménologiques sur les processus du vivant soit à la fois possible et féconde.

International audience; Comme d’autres champs de la connaissance, la biologie est maintenant affaire de big data. Nous souhaitons rendre compte ici d’une analyse réflexive que nous portons, comme chercheurs en biologie, sur les changements de pratiques et les représentations qui accompagnent l’ère génomique qui tend à réduire le vivant à de l’information. Nous développons l’idée que les technologies liées au séquençage massif induisent une relation modifiée au vivant et des approches expérimentales radicalement nouvelles. Pour nous-mêmes, et au delà de leur efficacité, nous percevons ces changements comme porteurs d’une crise qui pousse à rechercher hors du champ disciplinaire strict de la biologie, des ressources réflexives, notamment au voisinage de la pratique artistique ou d’une certaine biologie philosophique.

On lit de plus en plus a voix haute, dans les bibliotheques, les musees, les theâtres, les ecoles, les jardins. A partir des discours tenus par des lecteurs professionnels, nous proposons d’analyser les modalites tres particulieres de cette pratique de la lecture. En prenant conscience du caractere materiel des mots traversant son corps « de bas en haut », le lecteur reconcilie dans le meme mouvement ce corps et son esprit. A travers le souffle de sa voix, ce n’est pas le texte mais la vie du monde sensuel elle-meme qu’il presente aux auditeurs spectateurs.

International audience; Heterochromatin assembly in fission yeast relies on the processing of cognate noncoding RNAs by both the RNA interference and the exosome degradation pathways. Recent evidence indicates that splicing factors facilitate the cotranscriptional processing of centromeric transcripts into small interfering RNAs (siRNAs). In contrast, how the exosome contributes to heterochromatin assembly and whether it also relies upon splicing factors were unknown. We provide here evidence that fission yeast Spf30 is a splicing factor involved in the exosome pathway of heterochromatin silencing. Spf30 and Dis3, the main exosome RNase, colocalize at centromeric heterochromatin and euchromatic genes. At the centromeres, Dis3 helps recruiting Spf30, whose deficiency phenocopies the dis3-54 mutant: heterochromatin is impaired, as evidenced by reduced silencing and the accumulation of polyadenylated centromeric transcripts, but the production of siRNAs appears to be unaffected. Consistent with a direct role, Spf30 binds centromeric transcripts and locates at the centromeres in an RNA-dependent manner. We propose that Spf30, bound to nascent centromeric transcripts, perhaps with other splicing factors, assists their processing by the exosome. Splicing factor intercession may thus be a common feature of gene silencing pathways.

Regulation of poly(A) tail length during mRNA 3'-end formation requires a specific poly(A)-binding protein in addition to the cleavage/polyadenylation machinery. The mechanism that controls polyadenylation in mammals is well understood and involves the nuclear poly(A)-binding protein PABPN1. In contrast, poly(A) tail length regulation is poorly understood in yeast. Previous studies have suggested that the major cytoplasmic poly(A)-binding protein Pab1p acts as a length control factor in conjunction with the Pab1p-dependent poly(A) nuclease PAN, to regulate poly(A) tail length in an mRNA specific manner. In contrast, we recently showed that Nab2p regulates polyadenylation during de novo synthesis, and its nuclear location is more consistent with a role in 3'-end processing than that of cytoplasmic Pab1p. Here, we investigate whether PAN activity is required for de novo poly(A) tail synthesis. Components required for mRNA 3'-end formation were purified from wild-type and pan mutant cells. In both situations, 3'-end formation could be reconstituted whether Nab2p or Pab1p was used as the poly(A) tail length control factor. However, polyadenylation was more efficient and physiologically more relevant in the presence of Nab2p as opposed to Pab1p. Moreover, cell immunofluorescence studies confirmed that PAN subunits are localized in the cytoplasm which suggests that cytoplasmic Pab1p and PAN may act at a later stage in mRNA metabolism. Based on these findings, we propose that Nab2p is necessary and sufficient to regulate poly(A) tail length during de novo synthesis in yeast.

Eukaryotic RNA polymerase II transcribes precursors of mRNAs and of non-protein-coding RNAs such as snRNAs and snoRNAs. These RNAs have to be processed at their 3' ends to be functional. mRNAs are matured by cleavage and polyadenylation that require a well-characterized protein complex. Small RNAs are also subject to 3' end cleavage but are not polyadenylated. Here we show that two newly identified proteins, Pti1p and Ref2p, although they were found associated with the pre-mRNA 3' end processing complex, are essential for yeast snoRNA 3' end maturation. We also provide evidence that Pti1p probably acts by uncoupling cleavage and polyadenylation, and functions in coordination with the Nrd1p-dependent pathway for 3' end formation of non-polyadenylated transcripts.

International audience; Increasing evidence implicates cohesin in the control of gene expression. Here we report the first analysis of cohesin-dependent gene regulation in fission yeast. Global expression profiling of the mis4-367 cohesin loader mutant identified a small number of upregulated and downregulated genes within subtelomeric domains (SD). These 20- to 40-kb regions between chromosome arm euchromatin and telomere-proximal heterochromatin are characterized by a combination of euchromatin (methylated lysine 4 on histone H3/methylated Tysine 9 on histone H3 [H3K4me]) and heterochromatin (H3K9me) marks. We focused our analysis on the chromosome 1 right SD, which contains several upregulated genes and is bordered on the telomere-distal side by a pair of downregulated genes. We find that the expression changes in the SD also occur in a mutant of the cohesin core component Rad21. Remarkably, mutation of Rad21 results in the depletion of Swi6 binding in the SD. In fact, the Rad21 mutation phenocopied Swi6 loss of function: both mutations led to reduced cohesin binding, reduced H3K9me, and similar gene expression changes in the SD. In particular, expression of the gene pair bordering the SD was dependent both on cohesin and on Swi6. Our data indicate that cohesin participates in the setup of a subtelomeric heterochromatin domain and controls the expression of the genes residing in that domain.

International audience; Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.

SummarySister-chromatid cohesion is mediated by cohesin, a ring-shape complex made of four core subunits called Scc1, Scc3, Smc1, and Smc3 in Saccharomyces cerevisiae (Rad21, Psc3, Psm1, and Psm3 in Schizosaccharomyces pombe). How cohesin ensures cohesion is unknown, although its ring shape suggests that it may tether sister DNA strands by encircling them [1]. Cohesion establishment is a two-step process. Cohesin is loaded on chromosomes before replication and cohesion is subsequently established during S phase. In S. cerevisiae, cohesin loading requires a separate complex containing the Scc2 and Scc4 proteins. Cohesin rings fail to associate with chromatin and cohesion can not establish when Scc2 is impaired [2]. The mechanism of loading is unknown, although some data suggest that hydrolysis of ATP bound to Smc1/3 is required [3, 4]. Scc2 homologs exist in fission yeast (Mis4), Drosophila, Xenopus, and human [5–10]. By contrast, no homolog of Scc4 has been identified so far. We report here on the identification of fission yeast Ssl3 as a Scc4-like factor. Ssl3 is in complex with Mis4 and, as a bona fide loading factor, Ssl3 is required in G1 for cohesin binding to chromosomes but dispensable in G2 when cohesion is established. The discovery of a functional homolog of Scc4 indicates that the machinery of cohesin loading is conserved among eukaryotes.

Recent studies of mRNA export factors have provided additional evidence for a mechanistic link between mRNA 3'-end formation and nuclear export. Here, we identify Nab2p as a nuclear poly(A)-binding protein required for both poly(A) tail length control and nuclear export of mRNA. Loss of NAB2 expression leads to hyperadenylation and nuclear accumulation of poly(A)(+) RNA but, in contrast to mRNA export mutants, these defects can be uncoupled in a nab2 mutant strain. Previous studies have implicated the cytoplasmic poly(A) tail-binding protein Pab1p in poly(A) tail length control during polyadenylation. Although cells are viable in the absence of NAB2 expression when PAB1 is overexpressed, Pab1p fails to resolve the nab2Delta hyperadenylation defect even when Pab1p is tagged with a nuclear localization sequence and targeted to the nucleus. These results indicate that Nab2p is essential for poly(A) tail length control in vivo, and we demonstrate that Nab2p activates polyadenylation, while inhibiting hyperadenylation, in the absence of Pab1p in vitro. We propose that Nab2p provides an important link between the termination of mRNA polyadenylation and nuclear export.

Lipofectin, which is a mixture of neutral lipid with a cationic lipid, has been widely used to enhance cellular delivery of phosphorothioate, 2'-sugar-modified, and chimeric antisense oligonucleotides. Phosphodiester oligonucleotides delivered with Lipofectin usually do not elicit antisense activity probably because cationic lipid formulations do not sufficiently protect unmodified oligonucleotides from nuclease degradation. We show that a cationic polymer, polyethylenimine (PEI), improves the uptake and antisense activity of 3'-capped 20-mer and 12-mer antisense phosphodiester oligonucleotides (PO-ODN) targeted to different regions of Ha-ras mRNA and to the 3'-untranslated region (3'-UTR) of C-raf kinase. In contrast, PEI, which forms a very stable complex with the 20-mer phosphorothioate oligonucleotide (PS-ODN), does not enhance its antisense activity. Using fluorescently labeled carriers and ODN, we show that PEI-PS-ODN particles are very efficiently taken up by cells but PS-ODN is not dissociated from the carrier. Our results indicate that carrier-ODN particle size and stability and ODN release kinetics vary with the chemical nature of the ODN and the carrier being transfected into the cells. The very low cost of PEI compared with cytofectins and the increased affinity for target mRNA and decreased affinity for proteins of PO-ODN compared with PS-ODN make the use of PEI-PO-ODN very attractive.

Publisher Summary Antisense technology can knock out selected genes, including disease-causing genes in cultured cells. A number of first-generation antisense compounds have entered human clinical trials. Most of these oligonucleotides are nuclease-resistant phosphorothioates (PS). However, it has been recognized for some years that these compounds may have nonsequence-specific effects on cellular functions. Compared to phosphodiester oligonucleotides (PO), phosphorothioates have a decreased affinity for complementary RNA sequences and a generally increased affinity for cellular proteins. Usually, in cell culture experiments, oligonucleotides with a phosphorothioate backbone are delivered with a cationic lipid. Cationic lipids increase cellular uptake and facilitate nuclear accumulation of the oligonueleotides. Phosphodiester oligonucleotides are degraded rapidly by exo- and endonucleases. The stability of phosphodiester oligonucleotides can be increased by blocking the 3'-hydroxyl function. There are few examples in the literature relating the biological activity of phosphodiester oligonucleotides. It is commonly thought that even 3' end-protected phosphodiester oligonucleotides are not active as antisense because they are not stable enough within cells. Few studies, however, have investigated the efficacy of cationic lipids to transfect oligonucleotides bearing different backbone modifications. This chapter shows that the transfection efficacy of a carrier depends on the chemical form of the nucleic acid being transfected.

We have previously described the rational design of mutation-selective antisense oligonucleotides targeted to codon 12 of oncogenic Ha-ras mRNA. In order to further improve the biological efficacy of these unmodified oligonucleotides, we have studied three different classes of modifications: peptide nucleic acid backbone (PNA), sugar modification (2'-O-methyl) and phosphoramidate linkage (PN). We show that PNA is unique among the investigated steric blocking agents in its ability to specifically inhibit the translation of Ha-ras mRNA in vitro. The PNA-RNA hybrid (Tm=86 degrees C), which is not dissociated by cellular proteins and resists phenol extraction and urea denaturing conditions, specifically blocks the translation of mutated Ha-ras mRNA. A PNA tridecamer which forms with wild-type Ha-ras mRNA a duplex with a central mismatch had little effect on mRNA translation. Codon 12 is located close to the translation initiation site and hybridization of the PNA at this position may interfere with the assembly of the translation initiation complex. To test whether polypeptide chain elongation can also be blocked, we have targeted PNA tridecamers to codons in the 74, 128 and 149 regions. These PNAs form equally stable duplexes as that formed by the PNA targeted to the codon 12 region (ten G.C base-pairs out of 13). We show that PNA-RNA duplexes block the progression of the 80 S ribosome. Therefore, it is possible to arrest translation with concomitant production of a truncated protein by using duplex-forming PNA oligonucleotides targeted to a G+C-rich sequences. Our data demonstrate for the first time that a non-covalent duplex can arrest the translation machinery and polypeptide chain elongation.