|Number of pages||3|
|State||Published - 1 Apr 2016|
Bibliographical noteFunding Information:
Yaron Shav-Tal Yaron.Shav-Tal@biu.ac.il The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan 52900, Israel The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology Bar-Ilan University Ramat Gan 52900 Israel RNA is one of the pillars of the gene expression pathway. In these exciting times when new RNA species are frequently revealed, many of which are non-coding or of unexpected size or structure (e.g. circular RNAs), it is imperative to develop tools and methods for the detection of RNA molecules in all cell types. This special issue of METHODS is therefore dedicated to the ever-expanding world of RNA detection techniques that are applied in order to locate, track and quantify RNA in fixed or living cells, tissues and whole organisms. The development of new techniques ultimately drives the biological questions we are able to put forward, and as such, issues that once have seemed out of reach, suddenly become trivial to address. This is true for the RNA world. Over time it has become possible to examine not only “where” an RNA is located, but also “when” do transport and localization take place, and “how” does an RNA molecule travel from one location to another. This special issue attempts to give a taste of a variety of RNA visualization procedures and their application on different cell types. Several reviews provide a wider perspective of the field and how it has transformed over the years. In the opening article, Joe Gall  provides a personal historical reflection on in situ nucleic acid hybridization (ISH), a revolutionary technique in molecular cell biology that was pioneered by him, starting with his PhD research in the 1950s. At the time, it became apparent that DNA carries the genetic information. Gall studied giant chromosomes of insect salivary glands and amphibian oocytes, realizing that the cytological analysis of chromosome structure will lead to understanding the functions of these DNA molecules. He describes his pursuit of this information as new experimental techniques became available. In order to validate the hybridization approach he targeted amplified ribosomal DNA (rDNA) regions in Xenopus oocytes with a radioactive labeled probe generated from ribosomal RNA (rRNA). Gall not only delicately paints a picture of the scientific environment he was working in and the questions driving the path to the establishment of ISH, but also describes the many pitfalls that occurred along the way. As one of the reviewers of the paper has stated, the conceptual path of thinking on how to improve an experiment that did not necessarily work in the beginning, comes close to the “soul of the scientific method”, and should serve as a didactic tool for students treading through the tracks of experimental lab work. Amazingly, the color figures that appear in this article were taken from the original microscope slides that were initially published as black and white figures. Using hybridization of labeled nucleic acid probes to detect DNA and RNA has advanced in many directions using a variety of tagging molecules, but it is hard to imagine the field of nucleic acid detection without the fluorescent progression of ISH to FISH, and the contribution of studies by Robert Singer to make this technique widely available and useful for the detection of single molecules (reviewed elsewhere). DNA FISH is commonly used for labeling chromosomal regions, whereas RNA FISH is applied in numerous cell types for the detection of RNA species. Additional adaptations allow the combination of FISH with immunofluorescence so that RNA and protein are simultaneously detectable. Several articles in this special issue present the unique application of RNA FISH in a plethora of cells and organisms, the methods used to analyze the acquired images, and some original variations on the FISH technique that have developed over the years. The clear detection of single mRNA molecules in fixed cells using FISH, drove the field towards performing these experiments in living cells. Different techniques have been around for quite a while for tagging mRNA in vivo , and Lampasona and Czaplinski  describe the current approaches that have evolved for the tracking of mRNA in living cells using fluorescent RNA binding proteins. A main technique is the MS2-tagging system pioneered in Robert Singer’s laboratory, based on the insertion of a series of stem-loops into the mRNA of interest, thereby serving as a binding site for a fluorescent RNA-binding protein. Lampasona and Czaplinski lay out the questions that can be addressed using these tools, the technical considerations that should be taken into account, and how these should be applied in different cells and organisms. To complement the RNA-tagging techniques by fluorescent proteins, Holstein and Rentmeister  present the toolbox of click chemistry by which covalent labeling approaches introduce a small reactive group into the studied RNA, which can be applied in fixed cells and tissues. Ilgu et al.  discuss ‘‘light-up” aptamers (e.g. malachite green aptamer and Spinach) that cause an increase in fluorescence of their ligands when bound by the aptamer, and demonstrate how these can be used for the detection of RNA in vivo . A series of articles detail the actual detection of RNAs in either fixed or living cells and organisms. In some organisms, elegant genetic tools can be utilized to examine tagged RNAs encoded from their endogenous genomic location, while in some cell types exogenous gene constructs expressing the tagged RNAs are typically used. The physiological process of “RNA localization” has been identified in many cell types and organisms. Mechanisms of RNA localization are typically used by the organism for determining expression patterns in developing tissues and embryos. Abbaszadeh and Gavis  present two methods for examining RNA localization in fixed and living Drosophila oocytes and embryo. They describe the use of single molecule FISH (smFISH), a recent development of the RNA FISH method that can be easily applied for detecting endogenous mRNAs. Bolková and Lanctôt  demonstrate the improved application of smFISH in the worm model organism Caenorhabditis elegans . Hauptmann et al.  illustrate how enzymatic in situ amplification methods can be used to detect dual mRNA FISH signals in zebrafish embryos. Powrie et al.  show how MS2-tagged RNA in live Xenopus oocytes can be used to study the dynamics of RNA localization, particularly during oogenesis. Bleckmann and Dresselhaus  demonstrate how RNA localization can be examined in plant tissues using a fluorescent whole-mount RNA in situ hybridization (F-WISH) protocol they established. They validate the applicability of the technique in visualizing the subcellular mRNA distribution in Arabidopsis ovules and developing seeds. RNA detection is also being put to good use in agriculture-related studies, for instance, for the examination of microorganisms that can cause diseases in both animals and plants. Kliot and Ghanim  review this topic bringing examples of the detection of viruses, bacteria and fungi RNA within host cells. Miorin et al.  show how Flavivirus RNA can be detected in living cells, and demonstrate the use of photobleaching techniques in understanding the intra-cellular life cycle of this virus. Alonas et al.  show how multiply-labeled tetravalent RNA imaging probes (MTRIPs) can be used to track the genomic RNA of respiratory syncytial virus (RSV) RNAs in living cells. Kannaiah and Amster-Choder  review the use of these techniques for examining RNA in bacteria, as it has become apparent that even in bacteria that have one cell compartment, mechanisms of specific RNA localization are at play. Bensidoun et al.  show how RNA detection and tracking in vivo in the yeast Saccharomyces cerevisiae can teach us about an important step of gene expression, namely, nuclear-cytoplasmic mRNA export through the nuclear pore complex. Urbanek and Krzyzosiak  explain the application of RNA FISH in the detection of toxic nuclear RNA foci that appear in certain diseases, among others myotonic dystrophy or Huntington’s disease, resulting from the expansion of CAG and CUG repeats occurring within different genes. Wrapping up this series of articles showing the applicable sides of RNA detection, Lee et al.  show how smFISH can be used along with immunofluorescence for detecting RNA in two types of large, multinucleate cell types, the fungus Ashbya gossypii and cultured mouse myotubes. They also present a semi-automated image processing tool that systematically detects mRNAs in the smFISH images and statistically analyzes the spatial distribution of mRNAs. The analysis of the images acquired from fixed cells or frames from time-lapse movies requires the continuous development and improvement of detection and quantification approaches. Halpern and Itzkovitz  describe an smFISH-based technique and software tool (TransQuant) generated for measuring transcription and degradation rates in intact mammalian tissues, and which is based on dual-color FISH probe libraries that target introns and exons of the genes of interest. This approach can be used on intact mammalian tissues. Rino et al.  describe a software tool that they generated (STaQTool) to be used for the automatic tracking and quantification of fluorescence at the site of transcription in 3D stacks obtained from movies of live human cells. This method can be applied for the fluorescence detection of introns and nascent transcripts undergoing the splicing process in real-time. Finally, and in light of the imaging capabilities that are racing forward with development of super-resolution microscopy, two articles discuss RNA detection at super-resolution. Larkin and Cook  describe methods for measuring the relative distances between transcripts in cultured cells with a precision of a few tens of nanometers and performed on a standard fluorescence microscope. Mito et al.  demonstrate the application of multi-color fluorescent FISH techniques for the simultaneous detection of RNA and proteins using super-resolution microscopy (structured illumination microscopy, SIM). Altogether, the biological understanding of RNA location, localization and dynamics by use of state-of-the-art equipment and computerized analyses, will lead to functional studies to be routinely performed in cells and organisms, culminating in better and in depth understanding of the processes of gene expression. Last and not least, this is the time thank the many authors contributing to this special issue of METHODS for sharing their protocols, expertise, ideas, and views on the field. I am grateful to Dr. Lynne Maquat for recruiting me to this endeavor, and to the Managing Editor, Tiffany Hicks, for her everlasting patience and assistance. I hope that the information provided within will stimulate many more experiments and unravel many more mysteries of the RNA world. This work in my laboratory is supported by the Israel Science Foundation (ISF), the United States – Israel Binational Science Foundation (BSF) and the Gassner Fund for Medical Research .