Prion-like RNA binding proteins (RBPs) such as TDP43 and FUS are largely soluble in the nucleus but form solid pathological aggregates when mislocalized to the cytoplasm. What keeps these proteins soluble in the nucleus and promotes aggregation in the cytoplasm is still unknown. We report here that RNA critically regulates the phase behavior of prion-like RBPs. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro. Reduction of nuclear RNA levels or genetic ablation of RNA binding causes excessive phase separation and the formation of cytotoxic solid-like assemblies in cells. We propose that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble. Changes in RNA levels or RNA binding abilities of RBPs cause aberrant phase transitions.

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication, and protect, repair and restart damaged forks. Here we identify DONSON as a novel fork protection factor, and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilises forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATR-dependent signalling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity, and potentiating chromosomal instability. Hypomorphic mutations substantially reduce DONSON protein levels and impair fork stability in patient cells, consistent with defective DNA replication underlying the disease phenotype. In summary, we identify mutations in DONSON as a common cause of microcephalic dwarfism, and establish DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

Transcription factor-DNA interactions are life sustaining and therefore the subject of intensive research. In spite of vast effort, quantitative in vivo studies of the molecular mechanisms underlying these fundamental interactions remain challenging. In the preceding paper, we designed synthetic Sex combs reduced (Scr) peptides and validated genetically their function as transcriptional regulators. Here we present a controllable system for quantitative studies of protein-DNA interactions in live cells that enables us to "titrate" the concentration of the synthetic Scr peptides in a single cell. Using methods with single-molecule sensitivity, advanced fluorescence imaging and fluorescence correlation spectroscopy (FCS), we were able to study the kinetics of Scr-DNA interactions in live salivary gland cells, where Scr is normally expressed during development. We discerned freely moving Scr molecules, characterized the specific and nonspecific Scr peptide-DNA interactions, and estimated their corresponding dissociation constants (K d ) in vivo. Our results suggest that the synthetic Scr transcription factors find their specific target sites primarily by multiple association/dissociation events, the rapidity of which is largely owed to electrostatic interactions. Based on these new findings, we formulate a model mechanism and emulate the kinetics of Scr homeodomain-DNA interactions in live cells using numerical simulations.fluorescence correlation spectroscopy | single-molecule sensitivity | homeodomain | Sex combs reduced | synthetic peptides T ranscription factor-DNA interactions, the assembly of functional transcriptional complexes, and the mechanisms involved in target site recognition are central to understanding gene regulation in vivo. In spite of such relevance, kinetic studies of transcription factor-DNA interactions are limited and have been performed only for a handful of proteins. Most prominent studies include the lac repressor in Escherichia coli (1-4), nuclear receptor-directed transcription (5), helicase translocation along DNA (6), and restriction enzymes-DNA interactions (7,8). The majority of these studies are carried out with naked DNA or reconstituted chromatin, reflecting the in vivo situation only to a limited extent. Chromatin structure in live cells is highly dynamic, involving multiple interactions and transformations (9)(10)(11). Moreover, a number of cofactors and coactivators/corepressors are involved in transcriptional regulation, suggesting that gene expression is accomplished through a dynamic, spatiotemporally entangled interplay between concomitant processesprotein searching for specific target sites, rearrangements of DNA conformation, and recruitment of cofactors and/or coregulators (5, 12, 13). Such complexity is not easily mimicked in solution, therefore necessitating live cell experimentation. However, these measurements are complex, and kinetic studies of protein-DNA interactions in live cells remain scarce (3,4,14,15), mainly due to the limited number of experimental approach...