RNA at the surface of phase-separated condensates impacts their size and number

TLDR: In this article, a methodology to form RNA-containing condensates in living cells with controlled RNA and protein composition is presented, based on physical constraints, provided by RNAs localized on condensate surface.

Abstract: Membrane-less organelles, by localizing and regulating complex biochemical reactions, are ubiquitous functional subunits of intracellular organization. They include a variety of nuclear and cytoplasmic ribonucleoprotein (RNP) condensates, such as nucleoli, P-bodies, germ granules and stress granules. While is it now recognized that specific RNA and protein families are critical for the biogenesis of RNP condensates, how these molecular constituents determine condensate size and morphology is unknown. To circumvent the biochemical complexity of endogenous RNP condensates, the use of programmable tools to reconstitute condensate formation with minimal constituents can be instrumental. Here we report a methodology to form RNA-containing condensates in living cells with controlled RNA and protein composition. Our bioengineered condensates are made of ArtiGranule scaffolds undergoing liquid-liquid phase separation in cells and programmed to specifically recruit a unique RNA species. We found that RNAs localized on condensate surface, either as isolated RNA molecules or as a homogenous corona of RNA molecules around the condensate. This simplified system allowed us to demonstrate that the size of the condensates scales with RNA surface density, the higher the RNA density is, the smaller and more frequent the condensates are. Our observations suggest a mechanism based on physical constraints, provided by RNAs localized on condensate surface, that limit condensate growth and coalescence.

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RNA at the surface of phase-separated condensates
impacts their size and number
Audrey Cochard, Marina Garcia-Jove Navarro, Shunnichi Kashida, Michel
Kress, Dominique Weil, Zoher Gueroui
To cite this version:
Audrey Cochard, Marina Garcia-Jove Navarro, Shunnichi Kashida, Michel Kress, Dominique Weil,
et al.. RNA at the surface of phase-separated condensates impacts their size and number. 2021.
�hal-03371689�

1
RNA at the surface of phase-separated condensates impacts their
size and number
Audrey Cochard
1,2
, Marina Garcia-Jove Navarro
1
, Shunnichi Kashida
1
, Michel Kress
2
, Dominique
Weil
2
, Zoher Gueroui
1
*
1- PASTEUR, Department of Chemistry, École Normale Supérieure, PSL University, Sorbonne Université,
CNRS, 75005 Paris, France.
2- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine (IBPS), Laboratoire de Biologie du
Développement, F-75005 Paris, France.
*Correspondence: zoher.gueroui@ens.fr
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 22, 2021. ; https://doi.org/10.1101/2021.06.22.449254doi: bioRxiv preprint

2
Membrane-less organelles, by localizing and regulating complex biochemical reactions, are
ubiquitous functional subunits of intracellular organization. They include a variety of nuclear and
cytoplasmic ribonucleoprotein (RNP) condensates, such as nucleoli, P-bodies, germ granules and
stress granules. While is it now recognized that specific RNA and protein families are critical for the
biogenesis of RNP condensates, how these molecular constituents determine condensate size and
morphology is unknown. To circumvent the biochemical complexity of endogenous RNP
condensates, the use of programmable tools to reconstitute condensate formation with minimal
constituents can be instrumental. Here we report a methodology to form RNA-containing condensates
in living cells with controlled RNA and protein composition. Our bioengineered condensates are made
of ArtiGranule scaffolds undergoing liquid-liquid phase separation in cells and programmed to
specifically recruit a unique RNA species. We found that RNAs localized on condensate surface,
either as isolated RNA molecules or as a homogenous corona of RNA molecules around the
condensate. This simplified system allowed us to demonstrate that the size of the condensates scales
with RNA surface density, the higher the RNA density is, the smaller and more frequent the
condensates are. Our observations suggest a mechanism based on physical constraints, provided by
RNAs localized on condensate surface, that limit condensate growth and coalescence.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 22, 2021. ; https://doi.org/10.1101/2021.06.22.449254doi: bioRxiv preprint

3
It is increasingly recognized that biomolecular condensates contribute to organize cellular
biochemistry by concentrating and compartmentalizing proteins and nucleic acids. They include a
broad range of nuclear and cytoplasmic ribonucleoprotein (RNP) granules, such as nucleoli, P-bodies,
germ granules and stress granules. Remarkably, abnormal condensate maturation into toxic
aggregates is linked to viral infection, cancer, and neurodegenerative diseases
1
. Cellular condensates
harbour a large diversity in terms of biochemical compositions as well as functions. Nevertheless, a
unified model of formation via liquid-liquid phase separation (LLPS), where RNP constituents
interact through multivalent and weak interactions, has been proposed to understand their biogenesis
2–6
. In addition to their diverse compositions and functions, condensates are also diverse in size.
Whereas P-bodies or PML bodies are often diffraction-limited puncta, other condensates such as germ
granules, centrosomes, and nucleoli can reach few micrometres in size
2,7–10
. What sets condensate
size and number in cells remains to be understood.
Mounting evidence based on in vitro reconstitutions and cellular approaches underlined the
importance of multivalent interactions between RBPs and RNAs in shaping condensate biogenesis
and morphology. In particular, RNA molecules have been proven to play fundamental roles in
determining the structure, dynamic and biophysical properties of condensates
11
. For instance, RNAs
act as molecular seeds to nucleate phase separated-condensates and regulate their assembly in a
spatiotemporal manner
12–17
. On the opposite, high RNA concentration can dissolve condensates and
keep prion-like RBPs soluble in cell nucleus
18,19
. In addition to their formation or dissolution, RNA
molecules can also impact the viscosity of the RNP condensates as well as the dynamics of their
components in a sequence-dependent manner
20–22
. The different structures of RNAs can determine
the molecular specificity of RNP condensates and thus explain the coexistence of separate
condensates with distinct molecular compositions
23
. Moreover, RNA-RNA interactions between
unstructured RNAs can lead to the formation of non-spherical condensates
24
. Finally, RNAs can take
part in RNA-RBP interactions that drive the formation of multiphasic condensates, whose structure
relies on RNA concentration and on RNA-RBP interaction strength
22,25,26
. In addition to the
contribution of condensate constituents, extrinsic factors such as membrane, cytoskeleton and
chromatin can modulate LLPS and condensate biogenesis and coarsening
27–29
.
Interestingly, the biochemical and structural heterogeneity at the surface of condensates could
also influence their stability. For instance in C.elegans, the adsorption of MEG-3 on PGL droplets
drives the formation of a gel-like shell around a liquid core that eventually can stabilize P granules
and trap RNAs
30,31
. Moreover, using an artificial scaffold in cells named ArtiGranule (ArtiG), we
previously demonstrated that the condensation of the RNA-binding domain of the P-body protein
Pumilio was sufficient to attract Pumilio RNA targets and P-body proteins at the surface of the
condensates, which in turn impacted the seeding and the size of the condensates
32
. We therefore
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 22, 2021. ; https://doi.org/10.1101/2021.06.22.449254doi: bioRxiv preprint

4
hypothesized that the size selection of the condensates relies on the adsorption of RNP elements at
their surface, which may contribute to limit coarsening by steric exclusion
32
. However, extracting the
specific role of RNA on condensate morphology is still inaccessible since natural RNP condensates
emerge from a complex combination of RNA-protein, RNA-RNA, and protein-protein interactions
33–
35
. To reduce this complexity, we developed a methodology to reconstitute the formation of RNA-
containing condensates in living cells with controlled RNA and protein composition. Our
bioengineered condensates were constituted of ArtiG scaffolds undergoing LLPS in cells, which were
programmed to specifically recruit a unique RNA species. We first fused ArtiG scaffolds to an
orthogonal RNA binding domain chosen to interact specifically with a synthetic mRNA. We found
that RNAs localized on condensate surface, either as isolated RNA molecules or as a homogenous
corona of RNA molecules around the condensate. The ArtiG condensates remained distinct from
endogenous condensates, which enabled us to separate the role of the recruited RNA molecules from
other potential contributors. We first observed a negative correlation between the number of
condensates per cell and their mean diameter. By quantifying the localization and number of
individual RNA molecules, we additionally found that the higher the RNA density is, the smaller and
more numerous the condensates are. Overall, our data indicate that the size of RNP condensate scales
with RNA surface density, which can be explained by physical constraints limiting condensate growth
and coalescence.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 22, 2021. ; https://doi.org/10.1101/2021.06.22.449254doi: bioRxiv preprint

Frequently asked questions

Q1. What have the authors contributed in "Rna at the surface of phase-separated condensates impacts their size and number" ?

HAL this paper is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. 

Q2. What have the authors stated for future works in "Rna at the surface of phase-separated condensates impacts their size and number" ?

Not only its flexibility will enable to address other such basic biological issues in the future, but it could also be a mean to engineer novel properties within cells. 

Q3. What is the role of the client proteins in the formation of multidroplet systems?

Client proteins acting like surfactants may reduce the energy required for the formation of an interface between the dense and dilute phase and lead to size-conserved multidroplet systems instead of the expected single large condensed phase, with condensate size decreasing as a result of an increase in the client concentration. 

Q4. What are the main reasons for the dissolution of artificial condensates?

Controlled dissolution of artificial condensatesRecent studies suggest that the formation and stability of biological condensates are tightlyregulated by multiple stimuli, including post-translational modifications, biochemical reactions, or physical parameters such as temperature or osmotic pressure changes39,40. 

Q5. What is the role of RNAs in regulating the assembly of condensates?

For instance, RNAs act as molecular seeds to nucleate phase separated-condensates and regulate their assembly in a spatiotemporal manner12–17. 

Q6. What is the way to examine how condensates scale with RNA surface density?

The robust formation of ArtiG condensates in cells provides an efficient assay to examinebasic questions such as how condensate size scales with RNA surface density. 

Q7. How many RNA molecules were found on the surface of artig condensates?

The authors found that the RNA density at the surface of condensates was correlated to their size and number, with large condensates displaying only a few RNAs on their surface whereas high RNA density always implied smaller and more numerous condensates. 

Q8. How did the authors find that RNAs were localized on the condensate surface?

The authors found that RNAs localized on condensate surface, either as isolated RNA molecules or as a homogenous corona of RNA molecules around the condensate. 

Q9. How many mRNAs were recruited at the condensate surface?

When quantifying the total number of mRNA molecules dispersed in the cytoplasm and localized on ArtiGemGFP/MCP, the authors found that 34% ± 19% of the cytoplasmic mRNAs were specifically recruited at the condensate surface (mean of 430 recruited RNAs and 1200 dispersed RNAs per cell) (Fig. 1f). 

Q10. What is the effect of the RNA at the surface of ArtiG condensates?

Here the authors propose that the RNA present at the surface of ArtiG condensates cause a steric hindrance that may prevent the growth of condensates by both subunit addition and coalescence (Fig. 5d). 

Q11. What are the main reasons for the size of RNP condensates?

their data indicate that the size of RNP condensate scales with RNA surface density, which can be explained by physical constraints limiting condensate growth and coalescence. 

Q12. What is the role of RNA in the morphology of RNPs?

In this context, in vitro reconstitutions using purified components have been a powerful strategy to study the diverse roles of RNAs involved in specifying the structure, composition and dynamical properties of RNP granules11,22,25,45,46. 

Q13. What are the main roles of RNA molecules in shaping the structure, dynamic and biophysical properties?

In particular, RNA molecules have been proven to play fundamental roles in determining the structure, dynamic and biophysical properties of condensates11. 

Q14. What is the main reason why the use of motifs from RBPs could limit the understanding?

While these approaches provided a powerful mean to manipulate RNP condensate mimics in cells, the use of motifs from RBPs that are known to target thousands of RNA species could limit the understanding of observed effect in cells. 

Q15. What is the role of RNAs in shaping condensate biogenesis?

Mounting evidence based on in vitro reconstitutions and cellular approaches underlined theimportance of multivalent interactions between RBPs and RNAs in shaping condensate biogenesis and morphology.