Abstract: Organisms from bacteria to humans employ complex biological pathways powered by key biomacromolecules such as proteins and nucleic acids to coordinate essential cellular functions. Unfortunately, the function of a surprising number of biomacromolecules still lacks any clearly defined function. For example, disordered proteins and non-canonical nucleic acid structures such as G-quadruplexes (G4) are emerging cellular components whose importance, and cellular functions are just starting to become clear. Evidently, the regulatory and pathological actions of G4s are tightly controlled by cellular proteins, and associated with many disordered proteins. For example, G4 formation within expansion-prone repeats and their interaction with disordered proteins is linked to many human disorders. Therefore, the discovery of G4 binding proteins, understanding the mechanism of interaction, and determining how this leads to biomolecular condensation linking to human diseases is important but remains challenging to study. Using two independent high-throughput screening methods, we show SERF family proteins, originally discovered for their ability to accelerate amyloid formation,[1] specifically bind G4 in physiological conditions at low nanomolar affinity. G4s can affect gene regulation and suppress protein aggregation; however, whether and how these two activities are linked is still unknown. We find that G4 binding suppresses SERF's ability to promote protein aggregation.[2] We discovered SERF’s ribonucleoprotein function vital for stress granule establishment in vivo,[3] and confirmed SERF-G4 condensation in vitro. Further, RNAseq analysis shows SERF protein depletion impacts the mRNA abundance of genes that contain high G4 density and increases toxicity in motor neurons derived from ALS patients with mutations in the C9ORF72 gene that form intracellular RNA condensates. An array of biophysical and biochemical methods including NMR reveal in great detail the structure, function, and dynamic details of two human SERF proteins binding to G4.[2-4] Combining single-molecule microscopy, NMR, and large-scale simulation, we demonstrate protein-G4 condensate at high resolution, for perhaps the first time, how proteins and RNA interact within biomolecular condensates.[3] Our findings provide a structure-function paradigm to understand biomolecular condensates at high resolution and stipulate a molecular framework to understand disordered protein and G-quadruplex biology.

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