Nascent RNA Folding and RNP Assembly Revealed by Single-molecule Microscopy.

The regulation of gene expression requires many RNA-protein complexes, or ribonucleoproteins (RNPs). Understanding the molecular mechanisms guiding the assembly of RNPs is a crucial step towards characterizing their roles in the cell. Assembly of an RNP, like the ribosomal subunits or the spliceosome, often occurs on the fly as a major RNA component is synthesized and co-transcriptionally folded. Yet, the timing of transcription, RNA folding, and association of binding partners must be tightly coordinated to ensure proper and rapid RNP assembly. Single-molecule methods have been pivotal in uncovering the ways RNAs navigate folding landscapes during transcription and how binding partners have adapted to guide - or circumnavigate - the RNA folding pathway. In this review, we discuss several single-molecule imaging techniques that have been recently developed to address key questions related to the kinetic control of RNA folding during transcription, mechanisms driving recruitment of binding partners, and functional links between the transcription and RNP machineries that underlie the mechanism of RNP assembly. Finally, we provide insight into the next steps to advance applications of single-molecule methods to further our understanding of RNP assembly mechanisms across the domains of life.