Making the message clear: visualizing mRNA localization

Abstract

Localized mRNA provides spatial and temporal protein expression essential to cell development and physiology. To explore the mechanisms involved, considerable effort has been spent in establishing new and improved methods for visualizing mRNA. Here, we discuss how these techniques have extended our understanding of intracellular mRNA localization in a variety of organisms. In addition to increased ease and specificity of detection in fixed tissue, in situ hybridization methods now enable examination of mRNA distribution at the ultrastructural level with electron microscopy. Most significantly, methods for following the movement of mRNA in living cells are now in widespread use. These include the introduction of labeled transcripts by microinjection, hybridization based methods using labeled antisense probes and complementary transgenic methods for tagging endogenous mRNAs using bacteriophage components. These technical innovations are now being coupled with super-resolution light microscopy methods and promise to revolutionize our understanding of the dynamics and complexity of the molecular mechanism of mRNA localization.

Figure 1

Detecting RNA in fixed cells. (a) ISH on 5-μm-thick wax sections of Drosophila syncytial blastoderm embryos using a tritiated probe against fushi tarazu pair-rule transcripts that are expressed in seven stripes. Silver grains of the photographic emulsion were visualized using dark field microscopy. (b) ISH on a whole-mount Drosophila syncytial blastoderm using a DIG-labeled probe against transcripts encoding the Even-skipped pair-rule protein detected by an anti-DIG alkaline-phosphatase-coupled antibody highlighted by histochemical staining using NBT and X-phosphate. The dark histochemical precipitate was imaged in bright field and photographed with black-and-white photographic film. (c)Drosophila syncytial blastoderm treated as in (b). The shallow view shows expression stripes of nascent transcripts within nuclei. (Taken from I. Davis D.Phil. Thesis.)

Figure 2

Detecting RNA in fixed cells by fluorescence. (a) Indirect labeling of RNA through its association with ZBP1. Double indirect immuno-fluorescence of a fixed primary mouse embryo fibroblast shows that ZBP1 (mouse CRD-BP), pseudo-colored in red, localizes to the leading edge of a migrating cell. The actin cytoskeleton (green) is detected using a pan-actin antibody followed by an Alexa 488 labeled secondary antibody. Note that CRD-BP is excluded from the lamellipodia. The nucleus is stained with DAPI (blue) (courtesy of the Singer Lab). (b) Multiple fluorescence in situ hybridization (FISH) to compare the distribution of two RNA species. Primary chick embryo fibroblast cells were transfected with an eGFP-β-actin DNA plasmid containing the full-length human β-actin 3′ UTR followed by 24 MCP binding sites. The middle cell expresses human β-actin protein (blue) following successful transfection. The exogenous RNA (red) was detected specifically by FISH using probes against the MS2 stem-loops. Endogenous β-actin mRNA (green) was also detected using FISH (courtesy of the Singer Lab). (c) Single molecule FISH in C. elegans N2 embryos showing individual elt-2 mRNAs (red dots). The nuclei are highlighted by DAPI staining of DNA (blue) (courtesy of Arjun Raj) .

Figure 3

Detection of RNA in live tissue. (a) Localization of multiple RNAs via injection. Drosophila syncytial blastoderm expressing nuclear GFP (blue) first injected with an Alexa Fluor 546-labeled runt RNA (red) and shortly after with an Alexa Fluor 488-labeled runt RNA (green). Both RNAs were injected into the same site (arrow). The image shows the first RNA is already apically localized, whereas the second is in transit (courtesy of Renald Delanoue). (b) Tracking individual β-actin mRNA molecules in vivo with MTRIPs in a human epithelial cell. Images were taken at 0.5 Hz for 5 min. Areas of interest (b′–b′″) show trajectories of single mRNA granules (courtesy of Philip Santangelo) .

Figure 4

Different options for endogenous tagging of mRNAs in vivo. UNAfold (by Stewart and Zuker (http://dinamelt.bioinfo.rpi.edu/download.php) predicted secondary structures for RNA stem–loop sequences which could be inserted into RNA constructs for labeling mRNA. Highlighted regions indicate nucleotides important for the binding specificity of the corresponding binding protein [82–94].

Figure I

ISH-IEM on ultrathin frozen sections.

Figure II

MS2-MCP labeling the life cycle of endogenous mRNA.

Figure III

Super-resolution imaging by SI on the Sedat OMX.