(A) RNA-binding proteins and exogenous RNA motifs. Care must be taken to prevent interference of the hybridization probe with the secondary structure of the target RNA and with any protein-binding motifs present. Hybridization probes benefit from the use of bright and stable synthetic dyes. As for RNA-binding proteins, other approaches based on (ii) split fluorescent proteins, (iii and iv) energy transfer or (v) dye intercalation aim to reduce the background signal by keeping the label in a non-fluorescent state before it is bound to the RNA. (i) Multiple dye molecules attached to a single probe help to increase the SNR and are particularly useful for long observations of an individual molecule. Anti-sense hybridization probes can be tailored to target an RNA sequence with high specificity, but they are not cell permeable. This approach enables control of the concentration of fluorescent molecules and the use of bright and stable synthetic dyes, but provides no specificity for a certain RNA species. (iv) Alternatively, native RNA-binding proteins (RBPs) can be labelled in vitro and delivered into the cell – e.g. (iii) Endogenous expression levels can be maintained if both the modified RNA sequence and the fluorescent tag are stably incorporated into the genome. Similarly, an aptamer sequence can freeze the synthetic, cell-permeable dye DFHBI in a fluorescent conformation to mimic the chromophore of GFP. To overcome this, fluorescent proteins can either be directed to the nucleus by coupling them with an NLS, or (ii) bipartite fluorescent proteins can be used. However, unbound fluorescent proteins, such as those used in the MS2, PP7 or λN systems, result in high unspecific background. (i) Binding motifs can be introduced into the native RNA strand in order to recruit fluorescent proteins (FPs) or synthetic dye molecules to the target RNA. Combinations of dedicated imaging technology and in vivo labelling have enabled studies of nuclear export ( Grünwald and Singer, 2010 Siebrasse et al., 2012 Ma et al., 2013), and observation of the three-dimensional (3D) trajectories of single RNA particles within living eukaryotic cells ( Spille et al., 2015 Smith et al., 2015). Since then, labelling techniques, optical microscopy, tracking procedures and analysis tools have evolved dramatically (for review, see Weil et al., 2010 Eliscovich et al., 2013 Pitchiaya et al., 2014). The first successful labelling and imaging of single mRNPs in vivo was achieved about 10 years ago ( Fusco et al., 2003). Microinjection of mRNA that has been labelled in vitro with fluorescent nucleotides shows that different mechanisms can lead to distinct subcellular localization patterns of mRNPs, such as transport by the motor proteins kinesin, dynein and myosin ( Wilkie and Davis, 2001 Zimyanin et al., 2008 reviewed in Weil et al., 2010). In the early 1980s, transcripts were shown to localize non-randomly in Styela eggs and distribute differentially during embryogenesis ( Jeffery et al., 1983). 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. 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. 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. 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. 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. 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. RNA molecules carry out widely diverse functions in numerous different physiological processes in living cells.
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