RNA export through the nuclear pore complex: pathways, mechanisms, and imaging strategies.

Understanding how RNA molecules traverse the nuclear pore complex (NPC) is central to regulated gene expression because the NPC serves as the selective gateway for RNA export from the nucleus. Distinct RNA classes, including tRNAs, snRNAs, miRNAs, lncRNAs, piRNAs, rRNAs, mRNAs, and circRNAs, follow biogenesis-coupled pathways and engage specific transport receptors and accessory factors to cross this barrier. Recent advances in single-molecule and super-resolution microscopy now enable direct, NPC-resolved visualization of transport for selected RNA species, allowing quantitative measurements of export kinetics, 3D trajectories through the pore, and interaction dynamics with NPC substructures. Here, we review and compare what has been learned from NPC-resolved imaging across RNA classes studied to date, highlighting both shared organizing principles and cargo-specific behaviours. We then describe an experimental and analytical toolbox for NPC-resolved studies of mRNA and pre-ribosomal particle export, including RNA-labelling strategies, major single-molecule and super-resolution modalities, and quantitative metrics used to extract transport parameters. Finally, we discuss key technical and conceptual barriers that currently limit extension to other RNA classes, particularly small and/or low-abundance RNAs, and outline practical strategies to overcome these constraints. Together, this review provides a unified framework for the next generation of single-molecule dissection of RNA dynamics at the nuclear pore.