Regulation of Biomolecular Phase Separation by Post-Translational Modification

RNA binding proteins (RBPs) are major components of biomolecular condensates, and are of interest for their ability to undergo liquid-liquid phase separation (LLPS). Interestingly, the ability of many RBPs to undergo phase transition is regulated by specific post-translational modifications (PTM). CD BioSciences provides professional services to analyze the regulation of biomolecular condensates phase separation by PTM, focusing on the effects of PTM on the phase separation of RBPs in vitro and in cells.

Introduction

PTM is recognized as a key regulator of the assembly and properties of biomolecular condensates. Specifically, PTM can modulate the structure, charge, hydrophobicity, and other properties of proteins involved in phase separation to promote or oppose phase separation under different circumstances, thereby tuning the emergent properties and overall function of condensates. RBPs, as important components of ribonucleoprotein (RNP) granules (a dynamic membrane-free organelle formed via LLPS), play an important role in the control of cellular RNA metabolism role in the control of cellular RNA metabolism. However, RBPs are influenced by PTM and constantly adjust their activities to maintain cellular homeostasis. Of interest is that dysregulation of PTM in RBPs is associated with the pathophysiology of many different diseases. Aberrant PTM patterns can lead to distortions in the physiological role of RBPs and/or accelerated or disrupted degradation due to mislocalization, loss or gain of function.

Fig. 1. Examples of PTM-mediated regulation of RBPs. (Velázquez-Cruz A, et al., 2021)

Customized Services

We offer comprehensive post-translational modification services in liquid-liquid phase separation to analyze the regulation of LLPS by various PTMs to elucidate how PTMs control the assembly and properties of condensates, including phosphorylation, acetylation, poly (ADP-ribosyl)-ation (PAR), ubiquitination and methylation, etc. Here, our experts focus on PTM of RBPs because these modifications can affect the phase separation behavior, physiological function and pathological behavior of RBPs. We can help you analyze the key features of RBPs that are significantly altered by PTM, including but not limited to:

• Subcellular distribution of RBPs.
• Interaction of RBPs with their cognate transcripts and other RNA-associated proteins.
• The turnover rate of RBPs.
• The ability of RBPs to recruit biomolecular condensates.

Since RBPs are closely linked to cell viability and proper function, their dysregulation is involved in the etiology and/or pathogenesis of multiple diseases. We study the PTM of RBPs in disease pathophysiology to attempt drug development with a special focus on neurodegenerative diseases and cancer.

In addition, we provide in vitro and intracellular sedimentation assays to analyze the effect of specific PTMs on the solubility of RBPs as a readout of the phase separation of RBPs in vitro and in cells.

• Sedimentation assay to assess the effect of PTMs on phase separation of RBPs in vitro
  This method allows a direct side-by-side comparison of the phase separation of unmodified and modified RBPs in vitro under the same experimental conditions. Protease-mediated removal of the solubility label was followed by centrifugation after a short incubation period to separate the condensate from the supernatant. RBPs contents in the condensate and supernatant was subsequently quantified by SDS-PAGE and Western blot analysis.
• Sedimentation assay to assess the effect of PTMs on the phase separation of RBPs in cells
  This method is suitable for analyzing the effect of the same PTM on the solubility of RBPs in cells, in which wild-type proteins can be expressed versus protein variants with PTM-mimicking mutations and biochemically separated.

PTM of RBPs is an important regulator of ribonucleoprotein particle dynamics. We offer specialized services to analyze how PTM regulates phase separation and RNP granules dynamics, particularly arginine (Arg)-methylation and phosphorylation. Studying the PTM regulation of RBPs allows for a better understanding of pathophysiological processes. We aim to aid in the diagnosis, prognosis and treatment of many serious diseases by revealing new biomarkers and therapeutic targets. If you have any special requirements for our services, please feel free to contact us. We are looking forward to working together with your attractive projects.