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Methods for spatial and temporal imaging of the different steps involved in RNA processing at single‐molecule resolution

RNA plays a quintessential role as a messenger of information from genotype (DNA) to phenotype (proteins), as well as acts as a regulatory molecule (noncoding RNAs). All steps in the journey of RNA from synthesis (transcription), splicing, transport, localization, translation, to its eventual degradation, comprise important steps in gene expression, thereby controlling the fate of the cell. This lifecycle refers to the majority of RNAs (primarily mRNAs), but not other RNAs such as tRNAs. Imaging these processes in fixed cells and in live cells has been an important tool in developing an understanding of the regulatory steps in RNAs journey. Single‐cell and single‐molecule imaging techniques enable a much deeper understanding of cellular biology, which is not possible with bulk studies involving RNA isolated from a large pool of cells. Classic techniques, such as fluorescence in situ hybridization (FISH), as well as more recent aptamer‐based approaches, have provided detailed insights into RNA localization, and have helped to predict the functions carried out by many RNA species. However, there are still certain processing steps that await high‐resolution imaging, which is an exciting and upcoming area of research. In this review, we will discuss the methods that have revolutionized single‐molecule resolution imaging in general, the steps of RNA processing in which these methods have been used, and new emerging technologies.