Smart 3D super-resolution microscopy reveals the architecture of the RNA scaffold in a nuclear body

Abstract Small subcellular organelles orchestrate key cellular functions. How biomolecules are spatially organized within these assemblies is poorly understood. Here, we report an automated super- resolution imaging and analysis workflow that integrates confocal microscopy, morphological object screening, targeted 3D super-resolution STED microscopy and quantitative image analysis. Using this smart microscopy workflow, we targeted the 3D organization of an architectural RNA that constitutes the structural backbone of paraspeckles, a membraneless nuclear organelle. Using site-specific labeling, morphological sorting and particle averaging, we reconstructed the morphological space of paraspeckles along their development cycle from over 10,000 individual particles. Applying spherical harmonics analysis, we report so-far unknown heterotypes of RNA organization. By integrating multi- positional labeling, we determined the coarse conformation of the RNA within the organelle and found the 3’ end forming a loop-like structure at the surface of the paraspeckle. Our study reveals key structural features of nuclear paraspeckle structure and growth, as well as on the molecular organization of the scaffold RNA.