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Intracellular mRNA phase separation induced by cationic polymers for tumor immunotherapy.

ABSTRACT: The formation of biomolecular condensates via liquid‒liquid phase separation (LLPS) is an advantageous strategy for cells to organize their subcellular compartments for diverse functions. Recent findings suggest that RNA or RNA-related LLPS techniques have potential for the development of new cellular regulation strategies. However, manipulating RNA LLPS in living cells has great challenges. Herein, we report that cationic polymers (CPs) have strong RNA LLPS-inducing activity. By introducing CPs into living cells or RNA solutions, significant RNA LLPS was verified through confocal imaging, turbidity assays, and fluorescence recovery after photobleaching (FRAP) tests. Among them, turbidity kinetics determinations indicated that the hydrophilic positively charged amino groups on the CPs play essential roles in RNA phase separation. Moreover, the LLPS induced by the cationic polymers dramatically changed the gene expression patterns in the cells. Interestingly, we found that TGFβ1 mRNA was highly encapsulated in the RNA droplets, which lowered the immunosuppressive capability of the tumor cells and triggered marked antitumor reactions in a mouse breast cancer model. Thus, we present here the CP-based modulation of RNA LLPS as a novel transcriptional manipulation method with potential for cancer immunotherapy drug development.

SUBMITTER: Xing Z

PROVIDER: S-EPMC9548170 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Intracellular mRNA phase separation induced by cationic polymers for tumor immunotherapy.

Xing Zhen Z Xue Jing J Ma Xindian X Han Congwei C Wang Zhenzhen Z Luo Shunhuang S Wang Chunming C Dong Lei L Zhang Junfeng J

Journal of nanobiotechnology 20221008 1

The formation of biomolecular condensates via liquid‒liquid phase separation (LLPS) is an advantageous strategy for cells to organize their subcellular compartments for diverse functions. Recent findings suggest that RNA or RNA-related LLPS techniques have potential for the development of new cellular regulation strategies. However, manipulating RNA LLPS in living cells has great challenges. Herein, we report that cationic polymers (CPs) have strong RNA LLPS-inducing activity. By introducing CPs ...[more]

PMID: 36209170

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Project description:Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.

Project description:Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required to suppress phase separation decreases relative to longer polymers. Collectively, our results demonstrate that crowded, membrane-bound polymers are highly efficient suppressors of phase separation and suggest that the ability of lipid domains to resist steric pressure depends on both their lipid composition and the size and concentration of the membrane-bound polymers they incorporate.

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Intracellular mRNA phase separation induced by cationic polymers for tumor immunotherapy.

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Intracellular mRNA phase separation induced by cationic polymers for tumor immunotherapy.

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