RNA Phase Separation in Cancer: Investigating HSATII RNA folding and function

As the study of genome regulation is moving to understand the essential functions performed by long non-coding RNAs (lncRNAs), once written off as “junk”, and biophysical studies are revealing the role of phase-separated condensates in nuclear organization, Human Satellite II (HSATII) RNAs present an advantageous opportunity to synthesize these complementary approaches. HSATII DNA, found on the heterochromatic pericentromeres of many human chromosomes, is transcriptionally repressed in normal cells, but misregulation of these genomic regions in a variety of cancer cell lines allows for their aberrant transcription. These highly repetitive HSATII RNAs aggregate into large focal accumulations immediately adjacent to their sites of transcription and bind to gene expression regulatory proteins such as methyl-CpG binding protein 2, MeCP2. Here, I present evidence that HSATII RNAs possess folded secondary structures which allow them to self-assemble into spherical droplets via liquid-liquid phase separation in vitro. Further, I show how these structures can be disrupted in vivo, providing a platform for testing key hypotheses regarding HSATII RNA droplet structure and their ability to sequester nuclear proteins such as MeCP2. Future work on HSATII RNA folding and their ability to form multivalent RNA-RNA, RNA-protein interactions in vivo will build upon a growing understanding of lncRNAs’ regulatory capacity and their role as organizers of liquid-like nuclear compartments

Polyelectrolyte Complexation of Oligonucleotides by Charged Hydrophobic – Neutral Hydrophilic Block Polymers

Polyelectrolyte complex micelles (PCMs, core-shell nanoparticles formed by complexation of a polyelectrolyte with a polyelectrolyte-hydrophilic neutral block polymer) offer an attractive solution to the critical problem of delivering therapeutic nucleic acids, but few structure-property studies have been carried out to date. We present data comparing oligonucleotide PCMs formed with poly(vinylbenzyl trimethylammonium) as the cationic block to those using poly(lysine), which is more commonly used. Despite its higher charge density, increased hydrophobicity, and permanent charge, pVBTMA appears to complex DNA more weakly than does poly(lysine). Using small angle X-ray scattering and electron microscopy, we find that, at physiological ionic strength, PCMs formed from both cationic blocks exhibit very similar structure-property relationships, with PCM radius determined by the cationic block size and shape controlled by the hybridization state of the oligonucleotides. These observations narrow the design space for optimizing therapeutic PCMs and provide new insights into the rich polymer physics of polyelectrolyte self-assembly. <br /

RNA-Protein Phase Separation In Cancer: Investigating Human Satellite II RNA Structure And Function

Human Satellite II, a tandemly repeated stretch of DNA found near the centromeres of most human chromosomes, is not transcribed into RNA in normal human cells, but is expressed in many human cancer cell lines and tissues. HSATII transcripts accumulate in the nucleus adjacent to their sites of transcription and recruit nuclear regulatory proteins within these large nuclear foci. Recent evidence suggests that RNA and proteins within the nuclear environment can phase separate into liquid-like droplets, creating abberant structures, some of which are known to contribute to pathological effects. Here, we present evidence that HSATII RNA folds on itself to form defined secondary structures, which could enable HSATII RNA to participate in a variety of RNA-RNA or RNA-protein interactions. Indeed, we show that HSATII RNA phase separates into liquid-like droplets, a process that is dependent on the size and sequence of the individual transcripts. A dependence on length suggests that HSATII RNA phase separation requires either a certain complexity of secondary structure or minimum number of contacts between single molecules. We hypothesize that HSATII RNA droplets differentially solubilize specific proteins via RNA-protein interactions, which could explain the observed sequestration of genomic regulatory proteins to nuclear HSATII RNA accumulations. Phase separation offers a powerful lens through which to begin to understand how misregulation of transcription, via the creation of liquid-like droplets, can disrupt regulatory processes, whose slight alterations can tip the balance in favor of further misregulation

Polyelectrolyte Complexation of Oligonucleotides by Charged Hydrophobic—Neutral Hydrophilic Block Copolymers

Polyelectrolyte complex micelles (PCMs, core-shell nanoparticles formed by complexation of a polyelectrolyte with a polyelectrolyte-hydrophilic neutral block copolymer) offer a solution to the critical problem of delivering therapeutic nucleic acids, Despite this, few systematic studies have been conducted on how parameters such as polycation charge density, hydrophobicity, and choice of charged group influence PCM properties, despite evidence that these strongly influence the complexation behavior of polyelectrolyte homopolymers. In this article, we report a comparison of oligonucleotide PCMs and polyelectrolyte complexes formed by poly(lysine) and poly((vinylbenzyl) trimethylammonium) (PVBTMA), a styrenic polycation with comparatively higher charge density, increased hydrophobicity, and a permanent positive charge. All of these differences have been individually suggested to provide increased complex stability, but we find that PVBTMA in fact complexes oligonucleotides more weakly than does poly(lysine), as measured by stability versus added salt. Using small angle X-ray scattering and electron microscopy, we find that PCMs formed from both cationic blocks exhibit very similar structure-property relationships, with PCM radius determined by the cationic block size and shape controlled by the hybridization state of the oligonucleotides. These observations narrow the design space for optimizing therapeutic PCMs and provide new insights into the rich polymer physics of polyelectrolyte self-assembly

TDP-43 condensation properties specify its RNA-binding and regulatory repertoire

Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These “binding-region condensates” are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution

Self-Sorting and Coassembly of Fluorinated, Hydrogenated, and Hybrid Janus Dendrimers into Dendrimersomes

The modular synthesis of a library containing seven self-assembling amphiphilic Janus dendrimers is reported. Three of these molecules contain environmentally friendly chiral-racemic fluorinated dendrons in their hydrophobic part (<b>R</b>_<b>F</b>), one contains achiral hydrogenated dendrons (<b>R</b>_<b>H</b>), while one denoted hybrid Janus dendrimer, contains a combination of chiral-racemic fluorinated and achiral hydrogenated dendrons (<b>R</b>_<b>HF</b>) in its hydrophobic part. Two Janus dendrimers contain either chiral-racemic fluorinated dendrons and a green fluorescent dye conjugated to its hydrophilic part (<b>R</b>_<b>F</b><b>-NBD</b>) or achiral hydrogenated and a red fluorescent dye in its hydrophilic part (<b>R</b>_<b>H</b><b>-RhB</b>). These <b>R</b>_<b>F</b>, <b>R</b>_<b>H</b>, and <b>R</b>_<b>H</b>F Janus dendrimers self-assembled into unilamellar or onion-like soft vesicular dendrimersomes (DSs), with similar thicknesses to biological membranes by simple injection from ethanol solution into water or buffer. Since <b>R</b>_<b>F</b> and <b>R</b>_<b>H</b> dendrons are not miscible, <b>R</b>_<b>F</b><b>-NBD</b> and <b>R</b>_<b>H</b><b>-RhB</b> were employed to investigate by fluorescence microscopy the self-sorting and coassembly of <b>R</b>_<b>F</b> and <b>R</b>_<b>H</b> as well as of phospholipids into hybrid DSs mediated by the hybrid hydrogenated-fluorinated <b>R</b>_<b>HF</b> Janus dendrimer. The hybrid <b>R</b>_<b>HF</b> Janus dendrimer coassembled with both <b>R</b>_<b>F</b> and <b>R</b>_<b>H</b>. Three-component hybrid DSs containing <b>R</b>_<b>H</b>, <b>R</b>_<b>F</b>, and <b>R</b>_<b>HF</b> were formed when the proportion of <b>R</b>_<b>HF</b> was higher than 40%. With low concentration of <b>R</b>_<b>HF</b> and in its absence, <b>R</b>_<b>H</b> and <b>R</b>_<b>F</b> self-sorted into individual <b>R</b>_<b>H</b> or <b>R</b>_<b>F</b> DSs. Phospholipids were also coassembled with hybrid <b>R</b>_<b>HF</b> Janus dendrimers. The simple synthesis and self-assembly of DSs and hybrid DSs, their similar thickness with biological membranes and their imaging by fluorescence and ¹⁹F-MRI make them important tools for synthetic biology

Self-Sorting and Coassembly of Fluorinated, Hydrogenated, and Hybrid Janus Dendrimers into Dendrimersomes

The modular synthesis of a library containing seven self-assembling amphiphilic Janus dendrimers is reported. Three of these molecules contain environmentally friendly chiral-racemic fluorinated dendrons in their hydrophobic part (<b>R</b>_<b>F</b>), one contains achiral hydrogenated dendrons (<b>R</b>_<b>H</b>), while one denoted hybrid Janus dendrimer, contains a combination of chiral-racemic fluorinated and achiral hydrogenated dendrons (<b>R</b>_<b>HF</b>) in its hydrophobic part. Two Janus dendrimers contain either chiral-racemic fluorinated dendrons and a green fluorescent dye conjugated to its hydrophilic part (<b>R</b>_<b>F</b><b>-NBD</b>) or achiral hydrogenated and a red fluorescent dye in its hydrophilic part (<b>R</b>_<b>H</b><b>-RhB</b>). These <b>R</b>_<b>F</b>, <b>R</b>_<b>H</b>, and <b>R</b>_<b>H</b>F Janus dendrimers self-assembled into unilamellar or onion-like soft vesicular dendrimersomes (DSs), with similar thicknesses to biological membranes by simple injection from ethanol solution into water or buffer. Since <b>R</b>_<b>F</b> and <b>R</b>_<b>H</b> dendrons are not miscible, <b>R</b>_<b>F</b><b>-NBD</b> and <b>R</b>_<b>H</b><b>-RhB</b> were employed to investigate by fluorescence microscopy the self-sorting and coassembly of <b>R</b>_<b>F</b> and <b>R</b>_<b>H</b> as well as of phospholipids into hybrid DSs mediated by the hybrid hydrogenated-fluorinated <b>R</b>_<b>HF</b> Janus dendrimer. The hybrid <b>R</b>_<b>HF</b> Janus dendrimer coassembled with both <b>R</b>_<b>F</b> and <b>R</b>_<b>H</b>. Three-component hybrid DSs containing <b>R</b>_<b>H</b>, <b>R</b>_<b>F</b>, and <b>R</b>_<b>HF</b> were formed when the proportion of <b>R</b>_<b>HF</b> was higher than 40%. With low concentration of <b>R</b>_<b>HF</b> and in its absence, <b>R</b>_<b>H</b> and <b>R</b>_<b>F</b> self-sorted into individual <b>R</b>_<b>H</b> or <b>R</b>_<b>F</b> DSs. Phospholipids were also coassembled with hybrid <b>R</b>_<b>HF</b> Janus dendrimers. The simple synthesis and self-assembly of DSs and hybrid DSs, their similar thickness with biological membranes and their imaging by fluorescence and ¹⁹F-MRI make them important tools for synthetic biology