Labelling and imaging of single endogenous messenger RNA particlesin vivo

Abstract

RNA molecules carry out widely diverse functions in numerous different physiological processes in living cells. The RNA life cycle from transcription, through the processing of nascent RNA, to the regulatory function of non-coding RNA and cytoplasmic translation of messenger RNA has been studied extensively using biochemical and molecular biology techniques. In this Commentary, we highlight how single molecule imaging and particle tracking can yield further insight into the dynamics of RNA particles in living cells. In the past few years, a variety of bright and photo-stable labelling techniques have been developed to generate sufficient contrast for imaging of single endogenous RNAs in vivo. New imaging modalities allow determination of not only lateral but also axial positions with high precision within the cellular context, and across a wide range of specimen from yeast and bacteria to cultured cells, and even multicellular organisms or live animals. A whole range of methods to locate and track single particles, and to analyze trajectory data are available to yield detailed information about the kinetics of all parts of the RNA life cycle. Although the concepts presented are applicable to all types of RNA, we showcase here the wealth of information gained from in vivo imaging of single particles by discussing studies investigating dynamics of intranuclear trafficking, nuclear pore transport and cytoplasmic transport of endogenous messenger RNA.

Pre-ribosomal particles from nucleoli to cytoplasm

“…The mode of transport of these substrates within the nucleoplasm is constrained diffusion. Notably, RNA particles often exhibit phases of mobility, in which they diffuse as fast as theoretically expected for particles of a corresponding size in the effective nuclear viscosity, which is in the range of 3 to 5 mPas (reviewed by [ 36 ]. In general, single RNA tracking data from recent years confirm the Pederson lab’s early observations [ 17 ] that RNA particles spread evenly by constrained diffusion in the nuclei of cells within approximately 10 seconds.…”

Live Cell Imaging of Endogenous RNAs Using Pumilio Homology Domain Mutants: Principles and Applications

“…RNA probes for fluorescence live cell imaging have several requirements; the major requirements are specificity for the target RNA, detectability by fluorescence microscope observation, and the ability to be introduced into living cells effectively via a simple method. The two possible probe molecule designs that satisfy these requirements are nucleotide-based probes and protein-based probes. − …”

Long-term imaging of individual mRNA molecules in living cells

“…When co-expressed in the same cell, dimers of FP-fused coat proteins bind to each stem loop, enabling visualization of mRNAs and active transcription sites by fluorescence microscopy ( Braselmann et al., 2020 ; Fusco et al., 2003 ; Rath and Rentmeister, 2015 ; Sato et al., 2020 ; Tutucci et al., 2018a ; Vera et al., 2016 ). For most applications, coat proteins are also fused to a nuclear localization signal (NLS) to deplete unbound FP-MCP and -PCP from the cytoplasm, thereby increasing image contrast for mRNAs labeled in the cytoplasm ( Ben-Ari et al., 2010 ; Ferguson and Larson, 2013 ; Lenstra and Larson, 2016 ; Spille and Kubitscheck, 2015 ; Tutucci et al., 2018c ; Wu et al., 2012 ). Since the NLS on unbound FP-M/PCP will favor a nuclear localization, the reduced availability of free coat proteins will limit cycling of FPs on cytoplasmic mRNAs.…”