Nott et al. demonstrate that the disordered N-terminal region of Ddx4, a key component of nuage or germ granules, can form membraneless organelles both in vitro and in live cells. These organelles are stabilized by electrostatic interactions and are highly sensitive to temperature, ionic strength, arginine methylation, and splicing. The authors identify sequence determinants that are common in membraneless organelle proteins, suggesting that phase separation of disordered proteins is a general mechanism for forming regulated, membraneless organelles. The organelles provide an alternative solvent environment that can concentrate single-stranded DNA while excluding double-stranded DNA, indicating their role in localizing nucleic acids. The findings offer insights into the role of intrinsically disordered proteins in the spontaneous self-assembly of cellular membraneless organelles.Nott et al. demonstrate that the disordered N-terminal region of Ddx4, a key component of nuage or germ granules, can form membraneless organelles both in vitro and in live cells. These organelles are stabilized by electrostatic interactions and are highly sensitive to temperature, ionic strength, arginine methylation, and splicing. The authors identify sequence determinants that are common in membraneless organelle proteins, suggesting that phase separation of disordered proteins is a general mechanism for forming regulated, membraneless organelles. The organelles provide an alternative solvent environment that can concentrate single-stranded DNA while excluding double-stranded DNA, indicating their role in localizing nucleic acids. The findings offer insights into the role of intrinsically disordered proteins in the spontaneous self-assembly of cellular membraneless organelles.