Catégorie d'actualités : PASTEUR

Liquid–liquid phase separation is thought to be a key organizing principle in eukaryotic cells to generate highly concentrated dynamic assemblies, such as the RNP granules. Numerous in vitro approaches have validated this model, yet a missing aspect is to take into consideration the complex molecular mixture and promiscuous interactions found in vivo. Here we report the versatile scaffold ArtiG to generate concentration-dependent RNA–protein condensates within living cells, as a bottom-up approach to study the impact of co-segregated endogenous components on phase separation. We demonstrate that intracellular RNA seeds the nucleation of the condensates, as it provides molecular cues to locally coordinate the formation of endogenous high-order RNP assemblies. Interestingly, the co-segregation of intracellular components ultimately impacts the size of the phase-separated condensates. Thus, RNA arises as an architectural element that can influence the composition and the morphological outcome of the condensate phases in an intracellular context.

 

 

Press release available :
Building artificial organelles to examine the formation of RNA-protein compartments
(in french : Utiliser des organelles artificielles pour examiner les transitions de phase contrôlant la formation des compartiments ARN-protéines)

 

References
RNA is a critical element for the sizing and the composition of phase-separated RNA-protein condensates
Marina Garcia-Jove Navarro, Shunnichi Kashida, Racha Chouaib, Sylvie Souquere, Gerard Pierron, Dominique Weil, Zoher Gueroui
Nature Communications
DOI : 10.1038/s41467-019-11241-6

The animal-like cryptochrome of Chlamydomonas reinhardtii (CraCRY) is a recently discovered photoreceptor that controls the transcriptional profile and sexual life cycle of this alga by both blue and red light. CraCRY has the uncommon feature of efficient formation and longevity of the semireduced neutral form of its FAD cofactor upon blue light illumination. Tyrosine Y₃₇₃ plays a crucial role by elongating, as fourth member, the electron transfer (ET) chain found in most other cryptochromes and DNA photolyases, which comprises a conserved tryptophan triad. Here, we report the full mechanism of light-induced FADH^• formation in CraCRY using transient absorption spectroscopy from hundreds of femtoseconds to seconds. Electron transfer starts from ultrafast reduction of excited FAD to FAD^•– by the proximal tryptophan (0.4 ps) and is followed by delocalized migration of the produced WH^•+ radical along the tryptophan triad (∼4 and ∼50 ps). Oxidation of Y₃₇₃ by coupled ET to WH^•+ and deprotonation then proceeds in ∼800 ps, without any significant kinetic isotope effect, nor a pH effect between pH 6.5 and 9.0. The FAD^•–/Y₃₇₃^• pair is formed with high quantum yield (∼60%); its intrinsic decay by recombination is slow (∼50 ms), favoring reduction of Y₃₇₃^• by extrinsic agents and protonation of FAD^•– to form the long-lived, red-light absorbing FADH^• species. Possible mechanisms of tyrosine oxidation by ultrafast proton-coupled ET in CraCRY, a process about 40 times faster than the archetypal tyrosine-Z oxidation in photosystem II, are discussed in detail.

Reference:
Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome
Fabien Lacombat, Agathe Espagne, Nadia Dozova, Pascal Plaza,* Pavel Müller,* Klaus Brettel, Sophie Franz-Badur, Lars-Oliver Essen*
J. Am. Chem. Soc., 2019, 141, 13394-13409.
DOI: 10.1021/jacs.9b03680

Interactions between proteins play an essential role in metabolic and signaling pathways, cellular processes and organismal systems. We report the development of splitFAST, a fluorescence complementation system for the visualization of transient protein-protein interactions in living cells. Engineered from the fluorogenic reporter FAST (Fluorescence-Activating and absorption-Shifting Tag), which specifically and reversibly binds fluorogenic hydroxybenzylidene rhodanine (HBR) analogs, splitFAST displays rapid and reversible complementation, allowing the real-time visualization of both the formation and the dissociation of a protein assembly.

 

 

Press release available :

Spying on protein-protein interactions with fluorescent chemical-genetic hybrids

(in french : Espionner les interactions protéine-protéine avec des hybrides chemogénétiques fluorescents)

 

References:
A split fluorescent reporter with rapid and reversible complementation
Alison G. Tebo and Arnaud Gautier^*
PASTEUR, Département de chimie, École normale supérieure, PSL University, Sorbonne Université́, CNRS, 24 Rue Lhomond, 75005 Paris, France
Nature Communications 10, 2822 (2019) 
DOI : 10.1038/s41467-019-10855-0

The name of messenger RNAs-“messenger », has always implicated the role of this class of RNA molecules as to transmit DNA information to protein, an intermediate. But now our data suggest that an mRNA can function more. Using a bottom-up approach to combine magnetic nanoparticles, synthetic RNAs, and human cell extract, all confined in water-in-oil droplets, we observed that a messenger RNA not only can be translated into functional proteins, but can also serve as a spatiotemporal scaffold, to concentrate the nascent proteins and their function at the translation site, and for the time being of translation. This temporary site-specific concentration is observed to trigger structural polymorphism of actin filament meshworks, the choreographers of cell shape and function. From a biological perspective, this phenomenon may represent a general mechanism of spatial control that cells use to produce functional diversity.

 

 

References:
Nanoparticle-based local translation reveals mRNA as a translation-coupled scaffold with anchoring function
Shunnichi Kashida, Dan Ohtan Wang, Hirohide Saito, and Zoher Gueroui
PNAS 2019
https://doi.org/10.1073/pnas.1900310116

Flavoproteins often stabilize their flavin coenzyme by stacking interactions involving the isoalloxazine moiety of the flavin and an aromatic residue from the apoprotein. The bacterial FAD and folate-dependent tRNA methyltransferase TrmFO has the unique property of stabilizing its FAD coenzyme by an unusual H-bond-assisted π–π stacking interaction, involving a conserved tyrosine (Y₃₄₆ in Bacillus subtilis TrmFO, BsTrmFO), the isoalloxazine of FAD and the backbone of a catalytic cysteine (C₅₃). Here, the interaction between FAD and Y₃₄₆has been investigated by measuring the photoinduced flavin dynamics of BsTrmFO in the wild-type (WT) protein, C53A and several Y₃₄₆ mutants by ultrafast transient absorption spectroscopy. In C53A, the excited FAD very rapidly (0.43 ps) abstracts an electron from Y₃₄₆, yielding the FAD˙⁻/Y₃₄₆OH˙⁺ radical pair, while relaxation of the local environment (1.3 ps) of the excited flavin produces a slight Stokes shift of its stimulated emission band. The radical pair then decays via charge recombination, mostly in 3–4 ps, without any deprotonation of the Y₃₄₆OH˙⁺ radical. Presumably, the H-bond between Y₃₄₆ and the amide group of C₅₃ increases the pK_a of Y₃₄₆OH˙⁺ and slows down its deprotonation. The dynamics of WT BsTrmFO shows additional slow decay components (43 and 700 ps), absent in the C53A mutant, assigned to excited FAD_ox populations not undergoing fast photoreduction. Their presence is likely due to a more flexible structure of the WT protein, favored by the presence of C₅₃. Interestingly, mutations of Y₃₄₆ canceling its electron donating character lead to multiple slower quenching channels in the ps–ns regime. These channels are proposed to be due to electron abstraction either (i) from the adenine moiety of FAD, a distribution of the isoalloxazine–adenine distance in the absence of Y₃₄₆ explaining the multiexponential decay, or (ii) from the W₂₈₆ residue, possibly accounting for one of the decays. This work supports the idea that H-bond-assisted π–π stacking controls TrmFO’s active site dynamics, required for competent orientation of the reactive centers during catalysis.

 

 

Reference:
Ultrafast photoinduced flavin dynamics in the unusual active site of the tRNA methyltransferase TrmFO
Nadia Dozova, Fabien Lacombat, Charles Bou-Nader, Djemel Hamdane* and Pascal Plaza*
Phys. Chem. Chem. Phys., 2019, 21, 8743-8756
DOI: 10.1039/C8CP06072J