A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord1. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing2–4. Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies5,6, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function.

Summary 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.

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 (RF), one contains achiral hydrogenated dendrons (RH), while one denoted hybrid Janus dendrimer, contains a combination of chiral-racemic fluorinated and achiral hydrogenated dendrons (RHF) in its hydrophobic part. Two Janus dendrimers contain either chiral-racemic fluorinated dendrons and a green fluorescent dye conjugated to its hydrophilic part (RF-NBD) or achiral hydrogenated and a red fluorescent dye in its hydrophilic part (RH-RhB). These RF, RH, and RHF 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 RF and RH dendrons are not miscible, RF-NBD and RH-RhB were employed to investigate by fluorescence microscopy the self-sorting and co-assembly of RF and RH as well as of phospholipids into hybrid DSs mediated by the hybrid hydrogenated-fluorinated RHF Janus dendrimer. The hybrid RHF Janus dendrimer co-assembled with both RF and RH. Three-component hybrid DSs containing RH, RF, and RHF were formed when the proportion of RHF was higher than 40%. With low concentration of RHF and in its absence, RH and RF self-sorted into individual RH or RF DSs. Phospholipids were also co-assembled with hybrid RHF 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.

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.

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>