Single and Combined Methods to Specifically or Bulk-Purify RNA–Protein Complexes

The ribonome interconnects the proteome and the transcriptome. Specific biology is situated at this interface, which can be studied in bulk using omics approaches or specifically by targeting an individual protein or RNA species. In this review, we focus on both RNA- and ribonucleoprotein-(RNP) centric methods. These methods can be used to study the dynamics of the ribonome in response to a stimulus or to identify the proteins that interact with a specific RNA species. The purpose of this review is to provide and discuss an overview of strategies to cross-link RNA to proteins and the currently available RNA- and RNP-centric approaches to study RNPs. We elaborate on some major challenges common to most methods, involving RNP yield, purity and experimental cost. We identify the origin of these difficulties and propose to combine existing approaches to overcome these challenges. The solutions provided build on the recently developed organic phase separation protocols, such as Cross-Linked RNA eXtraction (XRNAX), orthogonal organic phase separation (OOPS) and Phenol-Toluol extraction (PTex).

Download Full-text

Phase transition of RNA−protein complexes into ordered hollow condensates

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922365117 ◽

2020 ◽

Vol 117 (27) ◽

pp. 15650-15658 ◽

Cited By ~ 8

Author(s):

Ibraheem Alshareedah ◽

Mahdi Muhammad Moosa ◽

Muralikrishna Raju ◽

Davit A. Potoyan ◽

Priya R. Banerjee

Keyword(s):

Phase Separation ◽

Protein Complexes ◽

Intrinsically Disordered Protein ◽

Liquid Droplets ◽

Isotropic Liquid ◽

Size Dependent ◽

Intrinsically Disordered ◽

Condensate Phase ◽

Rna Complexes ◽

Rna Protein Complexes

Liquid−liquid phase separation of multivalent intrinsically disordered protein−RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA−protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA−protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA−protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein−RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein−RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein−RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.

Download Full-text

Granule regulation by phase separation during Drosophila oogenesis

Emerging Topics in Life Sciences ◽

10.1042/etls20190155 ◽

2020 ◽

Vol 4 (3) ◽

pp. 355-364

Author(s):

M. Sankaranarayanan ◽

Timothy T. Weil

Keyword(s):

Phase Separation ◽

Translational Control ◽

Protein Complexes ◽

Physiological Role ◽

Drosophila Oogenesis ◽

And Function ◽

Rna Protein Complexes ◽

Liquid Liquid Phase Separation

Drosophila eggs are highly polarised cells that use RNA–protein complexes to regulate storage and translational control of maternal RNAs. Ribonucleoprotein granules are a class of biological condensates that form predominantly by intracellular phase separation. Despite extensive in vitro studies testing the physical principles regulating condensates, how phase separation translates to biological function remains largely unanswered. In this perspective, we discuss granules in Drosophila oogenesis as a model system for investigating the physiological role of phase separation. We review key maternal granules and their properties while highlighting ribonucleoprotein phase separation behaviours observed during development. Finally, we discuss how concepts and models from liquid–liquid phase separation could be used to test mechanisms underlying granule assembly, regulation and function in Drosophila oogenesis.

Download Full-text

Chromosome-associated RNA–protein complexes promote pairing of homologous chromosomes during meiosis in Schizosaccharomyces pombe

Nature Communications ◽

10.1038/s41467-019-13609-0 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 6

Author(s):

Da-Qiao Ding ◽

Kasumi Okamasa ◽

Yuki Katou ◽

Eriko Oya ◽

Jun-ichi Nakayama ◽

...

Keyword(s):

Phase Separation ◽

Long Noncoding Rna ◽

Noncoding Rna ◽

Rna Binding ◽

Rna Binding Proteins ◽

Protein Complexes ◽

Homologous Pairing ◽

Homologous Chromosomes ◽

Rna Protein Complexes ◽

Chromosomal Loci

AbstractPairing of homologous chromosomes in meiosis is essential for sexual reproduction. We have previously demonstrated that the fission yeast sme2 RNA, a meiosis-specific long noncoding RNA (lncRNA), accumulates at the sme2 chromosomal loci and mediates their robust pairing in meiosis. However, the mechanisms underlying lncRNA-mediated homologous pairing have remained elusive. In this study, we identify conserved RNA-binding proteins that are required for robust pairing of homologous chromosomes. These proteins accumulate mainly at the sme2 and two other chromosomal loci together with meiosis-specific lncRNAs transcribed from these loci. Remarkably, the chromosomal accumulation of these lncRNA–protein complexes is required for robust pairing. Moreover, the lncRNA–protein complexes exhibit phase separation properties, since 1,6-hexanediol treatment reversibly disassembled these complexes and disrupted the pairing of associated loci. We propose that lncRNA–protein complexes assembled at specific chromosomal loci mediate recognition and subsequent pairing of homologous chromosomes.

Download Full-text

Phase Transition of RNA-protein Complexes into Ordered Hollow Condensates

10.1101/2020.01.10.902353 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ibraheem Alshareedah ◽

Mahdi Muhammad Moosa ◽

Muralikrishna Raju ◽

Davit Potoyan ◽

Priya R. Banerjee

Keyword(s):

Phase Separation ◽

Protein Complexes ◽

Intrinsically Disordered Protein ◽

Liquid Droplets ◽

Stimuli Responsive ◽

Isotropic Liquid ◽

Intrinsically Disordered ◽

Heterotypic Interactions ◽

Rna Complexes ◽

Rna Protein Complexes

AbstractLiquid-liquid phase separation of multivalent intrinsically disordered protein-RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA-protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA-protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA-protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein-RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein-RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein-RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.Significance statementVesicular assemblies play crucial roles in subcellular organization as well as in biotechnological applications. Classically, the ability to form such assemblies were primarily assigned to lipids and lipid-like amphiphilic molecules. Here, we show that disordered RNA-protein complexes can form vesicle-like ordered assemblies at disproportionate mixture compositions. We also show that the ability to form vesicular assemblies is generic to multi-component systems where phase separation is driven by heterotypic interactions. We speculate that such vesicular assemblies play crucial roles in the formation of dynamic multi-layered subcellular membrane-less organelles and can be utilized to fabricate novel stimuli-responsive microscale systems.

Download Full-text

Alu RNA-protein complexes formed in vitro react with a novel lupus autoantibody.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)39098-1 ◽

1985 ◽

Vol 260 (21) ◽

pp. 11781-11786

Author(s):

R Kole ◽

L D Fresco ◽

J D Keene ◽

P L Cohen ◽

R A Eisenberg ◽

...

Keyword(s):

Protein Complexes ◽

Alu Rna ◽

Rna Protein Complexes

Download Full-text

Arabidopsis thaliana G3BP Ortholog Rescues Mammalian Stress Granule Phenotype across Kingdoms

International Journal of Molecular Sciences ◽

10.3390/ijms22126287 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6287

Author(s):

Hendrik Reuper ◽

Benjamin Götte ◽

Lucy Williams ◽

Timothy J. C. Tan ◽

Gerald M. McInerney ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Fusion Proteins ◽

Biological Function ◽

Protein Complexes ◽

Protein Family ◽

Knock Out ◽

Marker Proteins ◽

Interaction Partners ◽

Functional Analyses ◽

Rna Protein Complexes

Stress granules (SGs) are dynamic RNA–protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.

Download Full-text