Following the messenger: Recent innovations in live cell single molecule fluorescence imaging

Abstract

Messenger RNAs (mRNAs) convey genetic information from the DNA genome to proteins and thus lie at the heart of gene expression and regulation of all cellular activities. Live cell single molecule tracking tools enable the investigation of mRNA trafficking, translation and degradation within the complex environment of the cell and in real time. Over the last 5 years, nearly all tools within the mRNA tracking toolbox have been improved to achieve high‐quality multi‐color tracking in live cells. For example, the bacteriophage‐derived MS2‐MCP system has been improved to facilitate cloning and achieve better signal‐to‐noise ratio, while the newer PP7‐PCP system now allows for orthogonal tracking of a second mRNA or mRNA region. The coming of age of epitope‐tagging technologies, such as the SunTag, MoonTag and Frankenbody, enables monitoring the translation of single mRNA molecules. Furthermore, the portfolio of fluorogenic RNA aptamers has been expanded to improve cellular stability and achieve a higher fluorescence “turn‐on” signal upon fluorogen binding. Finally, microinjection‐based tools have been shown to be able to track multiple RNAs with only small fluorescent appendages and to track mRNAs together with their interacting partners. We systematically review and compare the advantages, disadvantages and demonstrated applications in discovering new RNA biology of this refined, expanding toolbox. Finally, we discuss developments expected in the near future based on the limitations of the current methods. This article is categorized under: RNA Export and Localization > RNA Localization RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes

Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles

“…These interactions stimulate the higher-order assembly of prion-like domains in protein misfolding diseases ( 32 ), together with disordered regions and RNA-scaffolded assembly ( 41 , 42 , 43 ). Additionally, structured protein domains are now emerging as mediators of widespread intracellular phase separation under conditions of high concentration and molecular crowding ( 44 , 45 ). Altered expression of RNA and protein components therefore can drastically influence both condensation and phase behavior itself ( Fig.…”

“…4. Single particle tracking (SPT) is a powerful tool to study dynamic recruitment of molecules to condensates ( 45 , 65 , 136 ). 5.…”

Fluorogenic aptamers resolve the flexibility of RNA junctions using orientation-dependent FRET

“…Foremost, genetically encodable fluorescent markers revolutionized proteomics, enabling the measurements of localization, folding, and interactions ( Rodriguez et al 2017 ). Further development of fluorescent tools to measure RNA folding, dynamics, and colocalization are required to keep pace with the ever-expanding field of RNA biology ( Schmidt et al 2020 ).…”

Studies on Viroid Shed Light on the Role of RNA Three-Dimensional Structural Motifs in RNA Trafficking in Plants

“…Currently, viroid RNA structures are mostly probed using in vitro assays. It will be beneficial to gain more insights into viroid structures at distinct subcellular and cellular compartments using in vivo probing methods, particularly those that can achieve observation at the single molecular level to detect the transient structural changes ( Stephenson et al, 2016 ; Yu and Lucks, 2019 ; Cawte et al, 2020 ; Schmidt et al, 2020 ).…”