RNA

An emerging concept over the past few years is that transcription and other cell signaling pathways are regulated by a diverse array of noncoding RNAs, such as microRNAs, long intergenic noncoding RNAs, Piwi-interacting RNAs, terminiassociated RNAs, and other classes of noncoding RNAs (Aravin et al. 2006; Han et al. 2007; Derrien et al. 2012). Our understanding of mRNA has also evolved. mRNA is no longer viewed as a simple intermediate between DNA and protein,

10.1 RNA Is Spatially and Temporally Regulated in Cells 267 10.2 The Need for RNA Imaging Technologies 269 10.3 Strategies to Detect RNA in Cells 270 10.4 A Strategy for Genetically Encoding Fluorescent RNA in Cells 271 10.5 Synthesis of GFP-Based Fluorophores 274 10.6 Identification of RNAs That Regulate the Fluorescence of a GFP-

Based Fluorophore 275 10.7 An RNA-Fluorophore Complex That Mimics Enhanced GFP 277 10.8 Imaging RNAs Using Spinach 279 10.9 Expanding the Spectral Diversity of RNA-Fluorophore

Complexes Using Novel Fluorophores 280 10.10 Spectral Tuning of RNA-Fluorophore Complexes 282 10.11 Considerations for Using Spinach to Image RNA in Live Cells 282 10.12 Imaging RNA in Multicellular Organisms 284 10.13 Strategies for Increasing Sensitivity of RNA Imaging 284 10.14 Future Directions 285 References 285

but instead is now known to be subjected to a wide range of posttranscriptional processing events, including splicing, nonsense-mediated decay, RNA editing, exo- and endonucleolytic degradation, adenosine methylation, polyadenylation, and deadenylation (Parker and Song 2004; Meyer et al. 2012). Another intriguing aspect of RNA biology is the finding that trinucleotide repeat-containing mRNAs exert specific gain-of-function toxicities associated with their accumulation in the nucleus and other intracellular sites (Ranum and Day 2004). In addition to these different regulatory pathways, recent studies indicate that RNAs transit through different parts of the cell as they undergo maturation to the final form needed in the cell. For example, nascent mRNA transcripts appear to be trafficked to specific intranuclear sites for processing events, such as splicing, nonsense-mediated decay, or for packaging into transport granules (Kiebler and Bassell 2006; Spector and Lamond 2011). After nuclear export, some RNAs have been localized to RNA-enriched intracellular structures including RNA granules, stress granules, and processing bodies (P-bodies) (Anderson and Kedersha 2002; Kiebler and Bassell 2006). The diversity of these RNA regulatory mechanisms makes it clear that RNA is regulated by a spatially complex and intricate network of regulatory mechanisms that have a critical role in gene expression.