A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat RNA

Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA.
Nature Methods (Impact Factor: 32.07). 10/2013; 10(12). DOI: 10.1038/nmeth.2701
Source: PubMed


Imaging RNA in living cells is a challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with Spinach, an 'RNA mimic of GFP'. We found that Spinach was dimmer than expected when used to tag constructs in living cells owing to a combination of thermal instability and a propensity for misfolding. Using systematic mutagenesis, we generated Spinach2 that overcomes these issues and can be used to image diverse RNAs. Using Spinach2, we detailed the dynamics of the CGG trinucleotide repeat-containing 'toxic RNA' associated with Fragile X-associated tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells.

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Available from: Rita L Strack, May 08, 2014
    • "Background suppression, similar to the split-fluorescent-protein approach, can be achieved if the protein-binding RNA sequence is replaced by an aptamer that recognizes small fluorescent ligands. For instance, the 'Spinach2' aptamer binds to the cell-permeable fluorophore 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and increases its quantum yield upon binding (Fig. 1) (Strack et al., 2013). The fluorophore is thus only detectable if bound to the target RNA. "
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    ABSTRACT: RNA molecules carry out widely diverse functions in numerous different physiological processes in living cells. The RNA lifecycle from transcription through 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 determining 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 localize and track single particles and analyse trajectory data are available to yield detailed information about the kinetics of all parts of the RNA lifecycle. While 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.
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    • "The resultant vectors are called pTRIC-Y and pTRIC-L (Y for tyrosine; L for leucine). To determine if pTRIC vectors can express heterologous tricRNAs, we inserted the Spinach2 RNA aptamer sequence (Strack et al. 2013), transfected the constructs into human cells, and carried out qRT-PCR analysis. Figure 3D shows that Spinach2 expression was readily detectable using divergent PCR primer pairs. "
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    ABSTRACT: We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating "designer" circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science. © 2015 Lu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
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    • "A major problem with using RNA aptamers is their poor folding in living cells. Aptamers are highly influenced by flanking sequences , which can interfere with aptamer folding (Martell et al., 2002; Strack et al., 2013). Although screening approaches have been described to improve aptamer folding (Martell et al., 2002), poor aptamer folding is a major roadblock that prevents their widespread use for diverse applications in living cells. "
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    ABSTRACT: RNA aptamers can be expressed in cells to influence and image cellular processes. Aptamer folding is maintained by inserting the aptamers into highly structured RNA scaffolds. Here, we show that commonly used RNA scaffolds exhibit unexpected instability and cleavage in bacterial and mammalian cells. Using an in-gel staining approach for rapid and simple detection of Spinach- or Broccoli-tagged RNAs in cells, we monitored the processing of RNAs tagged with scaffolded aptamers, revealing endonucleolytic cleavage, RNA instability, and poor expression. We reengineered a natural three-way junction structure to generate an alternative scaffold that enables stable aptamer expression in cells. This scaffold was used to create cassettes containing up to four Broccoli units, markedly enhancing the brightness of mammalian cells expressing cassette-tagged RNAs. These experiments describe methods for screening RNA cleavage events in cells and identify cell-compatible scaffolds that enable efficient tagging of RNAs with aptamers for cellular expression. Copyright © 2015 Elsevier Ltd. All rights reserved.
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