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Origins of life 

Fragments and bricks of living- Molecular and Structural Archeobiology- Heredity.

Context :

One remaining crucial point in the early life history is to understand how evolution passed from complex prebiotic chemistry to simple biology. Current cellular facts allow us to follow the link from chemical to biochemical metabolites, from the ancient to the modern world. Among the primeval organics we focus on the primordial nitrogenous bases scaffolding the initial darwinian ancestor (IDA). In this context, the “RNA world” hypothesis proposes that early in the evolution of life, RNA was responsible for the storage and transfer of genetic information and for the catalysis of biochemical reactions. The discovery of ribozymes at the beginning of the 80’s radically changed the field of molecular biology by revealing the existence of a new continent, the one of RNA – whose extraordinary biological properties have consistently been underlined since then –that could have been preceded by a more ancient RNA World. Several papers are devoted to these aspects of the RNA world (see The RNA World Cold Spring Harbor Lab edition). 

We contribute in this field mainly with the study of small ribozymes under extreme conditions (pressure, temperature) as well as with the discovery of a co-ribozyme selected in vitro (by SELEX), the Adenine-dependent hairpin ribozyme (ADHR). We also studied interactions between RNA and compartments as well as peptide-liposome interactions. Finally the persistance and the adaptability of viroids, that is of subcellular viruses considered as vestiges of ancient life, were shown in our laboratory. Along with the structural studies of ASBVd (Avocado Sunblotch Viroid) we showed the replication of this viroid in a non-photosynthetic eukaryote (yeast) as well as in a prokaryote (filamentous cyanobacterium Nostoc PCC7120) .

We also contribute for several years to the analysis of spatial meteorites, micrometeorites, dust and volatil (see publications).

Summary results:

Beyond the 4 canonical bases, over 100 modified nucleotides have been

identified on transfer RNAs and ribosomal RNAs. Several authors have proposed that modified nucleotides are relics of an ancient RNA world that was more diversified and more heterogenous than the contemporary world. The ancestor of nucleic acids (IDA: Initial Darwinian Ancestor) probably did not present a uniform structure and was possibly the result of co-polymerisation of different families.

In our search for such primitive building blocks, we noticed that N6-ribosyl -adenine, a compound easily synthesized under prebiotic conditions (Fuller, W.D., Sanchez, R.A and Orgel, L.E, 1972) has a free imidazole, a chemically reactive group, and is therefore a potential analog of the amino acid, histidine. We synthesized this compound and showed that it was indeed as active as histidine in the hydrolysis of paranitrophenyl acetate, a model substrate often used in studying enzymes catalyzing esterase-type reactions (Maurel and Ninio, 1987 ; Maurel and Convert, 1990, Maurel, 1991). Furthermore, numerous studies have shown that compounds such as N6 or N3-ribosyl-purine could have been essential links between the protein and the RNA worlds (Maurel, 1992).

It is also interesting to notice that some of the purine bases of nucleic acids were identified in the Murchison meteorite (Stoks and Schwartz, 1981 ; Callahan et al, 2011).

Pursuing this line of investigation we showed that nucleic acid-like polymers such as poly(allylamine)s incorporating N6-substituted adenine rings exhibit pronounced catalytic activities in the hydrolysis of PNPesters (Maurel et Décout, 1992, 1995 a and b ; Décout et Maurel, 1993). A coupled experimental and theoretical study allowed us to speculate on the origin of kinetic co-operativity in prebiotic catalysts (Ricard, Vergne, Décout et Maurel, 1996 ; Maurel et Ricard, 2006).

We have selected, using the SELEX (Systematic Evolution of Ligand by EXponential enrichment) method, RNA aptamers for adenine. Analysis of the affinity of the RNA aptamer for other purines reveals a mode of purine recognition involving weak hydrophobic stacking interactions with the entire purine ring and additional hydrogen bonding with the 6-NH2 group of adenine (Meli, Vergne, Décout et Maurel, 2002). Adenine and adenylated compounds play key roles in contemporary metabolisms and evidences exist that they played an important role in early evolution (Maurel et Décout, 1999; Meli, Albert-Fournier, Maurel, 2001). Cofactor assisted ribozymes could be considered as remnants of RNA world ribozymes handling exogenous prebiotic reactive ligands. RNAs binding specific cofactors, amino acids or analogs may have provided a kind of scaffold modeling early relationships between primeval tRNAs and specific reactive groups. They would have been pre-adaptors that led to the emergence of the modern genetic apparatus. 

We discovered a hairpin ribozyme strictly dependent on adenine (ADHR) as external co-factor for the reversible self-cleavage reaction. This co-ribozyme acts as a ribozyme using adenine as cofactor to perform a ribonuclease-type activity (Meli et al, 2002; 2003). 

Catalytic mechanism of the sTRSV (Satellite tobacco ringspot virus)  and CchMVd (Chrysanthemum Chlorotic Mottle Viroid) ribozymes and of the ADHR1 et ADHR2 mutants

Within the context of the RNA world, the study of the catalytic activity of a ribozyme under pressure makes it possible to examine the behavior of this molecule under extreme conditions. Today, many organisms are faced with such extreme conditions, but the mechanims and the behavior of these macromolecules are poorly understood. Our results on the wild ribozymes of sTRSV and CChMVd and on the mutants ADHR1 abd ADHR2 confirm the results obtained from cristallographic pictures and highlight the folding and plasticity of the RNA molecule. In addition, they provide informations on the amplitude of these dynamics (Tobé et al, 2005 ; Hervé et al, 2006, Buck et al, 2009 ; Ztouti et al, 2009 ; Kaddour et al, 2011 ; Kaddour et al, 2014). The activity, auto-association and the structure of the ribozymes ADHR1 and AHDR2 were studied by analytical centrifugation and neutron scattering. Stem-stem, loop-loop and stem-loop interactions play a major role in the regulation of the two ribozymes (Li et al, 2008). We followed the conformational changes of the pre- and post-catalytic states of ADHR1. Interaction of the RNA with adenine is transient, the dynamics induced constitutes the limiting step of the catalysis (Buck et al, 2009). 

In vitro selection of resistant RNA

   We are also interested in the study of the stability and conservation of RNA molecules. We have shown that high salt (Na) concentration plays a protective role in tRNA against thermal degradation allowing activity to be restored (Tehei et al, 2002). We selected thermo-halophilic aptamers in vitro that were resistant to degradation at 85°C in the presence of high salt for 65 h. The clones recovered were regrouped into two families (Family I and Family II) that each possesses its own physico-chemical characeristics as well as a secondary structure similar to that of numerous viroids.These two families correspond to two pathways leading to thermo-resistance. 

The molecular behavior thus highlighted allows us to define the survival limits in primitive (and/or extreme) conditions, and demonstrate that what is at stake for RNA molecules as also for the organisms during the course of evolution, is reproductive capacity (Vergne et al, 2006). Finally, we have characterized the behavior of ASBVd at high temperature in conditions close to that of hydrothermalism (El Murr et al, 2012).  

We have also developed a method to detect nucleic acids by SERS Raman spectrometry (Elamri et al, 2003, 2004, 2005) that allows us to detect nanomoles of oligonucleotides by selectively sounding constitutive adenyl residues. This method was used to study the kinetics of auto-cleavage of sub-nanomoles of ribozymes in solution (Percot et al, 2009).    

Cellularisation processes

Research studies in the field of the origins of cellular life need to pass by the comprehension of the assembly phenomena of compartmentalisation (membrane-related) structures starting from amphiphilic molecules.  

Such a line of investigation was initiated during my stay at The Salk Institute (1997) (Dr. L.E Orgel’s laboratory). We noticed that a sulfur-rich primitive atmosphere is consistent with astrophysical studies and must be taken into account for a sulfur-metabolizing step in prebiotic evolution. We performed prebiotic syntheses of peptides from thiolated amino acids and showed the direct oligomerization of thioglutamic acid without extrinsic activators, (Maurel and Orgel, 2000). We pursued the study of such reactions within a liposome and showed the lipid catalysis of thioglutamic acid oligomerization (Zepik et al, 2007). 

Field of applications

Therapeutic applications

The discovery of catalytic RNAs introduced the possibility of constructing therapeutic RNAs capable of performing trans-cleavage reactions. This type of ribozyme binds the substrates by base-pairing, cleaves the target RNA, and then liberates the cleavage products. With the aim of preventing the expression of two products of the Tpl2/cot gene, Proto-Cot and Cot, that are overexpressed in 40% of breast tumors, Yanli Li who did his Ph.D. thesis in our group, inserted into hairpin ribozymes, the sequence of 22 nucleotides from the Tpl2/cot gene. We caracterized these new ribozymes that are capable of cleaving the inserted fragment in cis and in trans (Li et al, 2007, 2008, 2009).    

Aptamers directed toward foetal cell markers

During gestation, the maternal organism, the foetus and the placenta are subjected to multiple reciprocal interactions (Maurel et Kanellopoulos, 2008).  The existence of cellular exchanges between the mother and the foetus are now well established, and the search of foetal male cells or of HLA antigens specific of the foetus are the two strategies used to detect cellular traffic. PCR and RT-PCR experiments performed on the sequences or the genes specific of the foetus or the mother - using murine models - have made it possible to establish that these events are frequent. Nevertheless, only the development of techniques allowing to reach extremely precise detection levels will lead to envisage non-invasive prenatal diagnostics so as to detect possible deficiencies or immunological incompatibilities. The aim of our studies performed in collaboration with the group of Colette Kanellopoulos consists in selecting foetal cells thanks to aptamers identified as specific markers of these cells. Such aptamers will allow affinity purification of the foetal cells, and after crosslink the caracterisation of the proteins recognized by the aptamers. 

At work :

   Viroids are the smallest known pathogenic agents. They are noncoding, single-stranded, closed-circular, “naked” RNAs, that replicate through RNA-RNA transcription. Viroids of the Avsunviroidae family possess a hammerhead ribozyme in their sequence, allowing self-cleavage during their replication. To date, viroids have only been detected in plant cells. We investigated the replication of Avocado sunblotch viroid (ASBVd) of the Avsunviroidae family in a nonconventional host, the yeast Saccharomyces cerevisiae. We demonstrated that ASBVd RNA strands of both polarities are able to self-cleave and to replicate in a unicellular eukaryote cell. We showed that the monomeric RNA of the viroid is destabilized by the nuclear 3’ and the cytoplasmic 5’ RNA degradation pathways. For the first time, our results provide evidence that viroids can replicate in other organisms than plants and that yeast contains all of the essential cellular elements for the replication of ASBVd (Delan-Forino et al, 2011). Furthermore the characteristics of each strand of ASBVd were analyzed using biophysical analysis techniques (Delan-Forino et al, 2014). ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in their thermal denaturation profiles. Subsequently, the secondary structures of ASBVd of plus and minus polarities were probed using the RNA-selective 2'-hydroxyl acylation method and analyzed by the primer extension (SHAPE) method. The models obtained show that ASBVd of the two polarities adopt different structures. Moreover, the results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in the in vivo viroid life cycle. 

In the same line of investigation we report the first Raman characterization of the structure and activity of ASBVd, for the plus and minus viroid strands. Both strands exhibit a typical A-type RNA conformation with an ordered double-helical content and a C3’-endo/anti sugar pucker configuration,  although small but specific differences are found in the sugar puckering and base-stacking regions. The ASBVd(-) is shown to self-cleave 3.5 times more actively than ASBVd(+). Deuteration and temperature increase perturb differently the double-helical content and the phosphodiester conformation, as revealed by corresponding characteristic Raman spectral changes. Our data suggest that the structure rigidity and stability are higher and the D2O accessibility to H-bonding network is lower for ASBVd(+) than for ASBVd(-). Remarkably, the Mg2+ activated self-cleavage of the viroid does not induce any significant alterations of the secondary viroid structure, as evidenced from the absence of intensity changes of Raman marker bands that, however exhibit small but noticeable frequency downshifts suggesting several minor changes in phosphodioxy, internal loops and hairpins of the cleaved viroids. Our results demonstrate the sensitivity of Raman spectroscopy in monitoring structural and conformational changes of the viroid and constitute the basis for further studies of its interactions with therapeutic agents and cell membranes (Hui Bon Hoa et al, 2014). 

 Viroids of the Asunviroidae family replicate in the chloroplasts of infected hosts. It is now largely admitted that chloroplasts evolved from a cyanobacterium ancestor. Because of this phylogeny relationship, we sought if a member of the Asunviroidae could be replicated in a cyanobacterium. We demonstrate that the Avocado sunblotch viroid (ASBVd) RNA strands of (-) and (+) polarities are able to replicate in the filamentous and heterocystous cyanobacterium Nostoc PCC7120. ASBVd replication does not impair the growth or the ability of the strain to sustain cell differentiation. Our results provide the first evidence that a prokaryotic cell possesses all the machinery required to sustain RNA replication without DNA intermediate (Latifi et al, 2014). These data are of great importance in our future knowledge on the origin and the evolution of viroids.

At the interface between Biology, Physics and Chemistry, we have presently underaken studies on the stucture, functions and interactions of nucleic acids. In one approach that is well established in physics, we wish to understand the laws that direct interactions in biological systems by performing experimental studies based on model systems and by modelisation based on concepts mainly developed in physics of soft matter. The second approach is based on the concept of the structure-function relationship in biology: rather than searching for general rules, the object is to understand specificity, that is, the differences in the structure selected by evolution, by performing experimental studies in physics based on several methods such as spectroscopy, diffraction, microscopy, etc. 

We are interested in the relationship, dynamics-function. At the molecular level, the activity of a biological system depends not only on the structural organisation of the system, but also on the movements that drive it. For certain activities such as catalysis, increased flexibility is required. In contrast, for others such as the transfer of electrons, a greater rigidity is essential. To explore the relationship, dynamics-function, original approaches are being introduced: a constant effective force (resilience) that can be measured by neutron diffusion, is strongly correlated with the biological function of systems. This method successfully validated on proteins of halophilic archeae demonstrates that adaptation to temperature results from forces that stabilize macromolecular structures and determine their flexibility in a specific scale of values. In collaboration with G. Zaccai at the ILL in Grenoble and A. Yonath of the Department of Structural Biology, Weizmann Institute  in Rehovoth, Israel, we perform dynamics measurements on a ribozyme, a viroid and on a "protoribosome" under different solvent and temperature conditions (Zaccai, G. Natali, F. Peters, J., Řihová, M., Zimmerman, E., Ollivier, J. Combet, J., Maurel, M-C., Bashan, A., & Yonath, A. (2016). The fluctuating ribosome: thermal molecular dynamics characterized by neutron scattering. Scientific Reports 6, 37138  doi:10.1038/srep37138).Developing and applying this type of methodological approach by spectrometry allowing to quantify functional intramolecular movements constitutes an important challenge that could open new areas of research. Our preliminary results suggest that during the course of evolution, selected RNAs presented specific dynamics that allowed structures and flexibility to be maintained within the narrow limits required for biological activity (Maurel et Leclerc, Elements, 2016 ; Steger, G., Riesner, D., Maurel M-C, Perreault, J-P., Viroids and Satellites. Oxford: Academic Press, 2017 ; Leclerc et al, Sc. Reports, 2016 ; Maurel et al, Viruses, 2019).   

Synthesis, evolution of bricks, fragments and small RNAs

We performed oligoribonucleotide synthesis and study whether the oligonucleotide itself could help to enhance its own synthesis, that is a ribozymic activity, with or without the help of lipids. This project was done in collaboration with Deamer’s laboratory (UCSC). Based on preliminary results, we have found that salts especially composed of ammonium can promote the polymerization under a simulated prebiotic environment (U.S. Provisional Patent Application for  “Non-enzymatic, salt-mediated synthesis of polynucleic acids” UC Case No.: SC 2013-946; PV No.: 482.36 (DJA); David Deamer, Marie-Christine Maurel, Laura Da Silva) and (Da Silva, L., Maurel, M-C., Deamer, D. J. Mol. Evol, 2015. Misuraca et al, Life, 2017)

Then we are working on prebiotic molecules as plausible vestiges (PRPP, nucleosides and nucleotides…) and performed their synthesis in prebiotic conditions (Akouche et al, Chemistry, 2016; Georgelin et al, Carbohydrate research, 2015; Akouche et al, Angewandte Chemie International, 2017; Pérez-Villa et al, The Journal of Physical Chemistry Letters, 2018) .

For More Recent works see Scientific Publications section.