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The ability to visualize RNA in situ is essential to dissect mechanisms for the temporal and spatial regulation of gene expression that drives development. Although considerable attention has been focused on transcriptional control, studies in model organisms like Drosophila have highlighted the importance of post-transcriptional mechanisms - most notably intracellular mRNA localization - in the formation and patterning of the body axes, specification of cell fates, and polarized cell functions. Our understanding of both types of regulation has been greatly advanced by technological innovations that enable a combination of highly quantitative and dynamic analysis of RNA. This review presents two methods, single molecule fluorescence in situ hybridization for high resolution quantitative RNA detection in fixed Drosophila oocytes and embryos and genetically encoded fluorescent RNA labeling for detection in live cells.

Intracellular mRNA localization critically contributes to proper brain development and function. A recent study demonstrates that mRNAs are actively transported in radial glial cells from the soma to the distal basal endfeet, where they are locally translated.

Intracellular mRNA localization is a conserved mechanism for spatially regulating protein production in polarized cells, such as neurons. The mRNA encoding the translational repressor Nanos (Nos) forms ribonucleoprotein (RNP) particles that are dendritically localized inDrosophilalarval class IV dendritic arborization (da) neurons. Innosmutants, class IV da neurons exhibit reduced dendritic branching complexity, which is rescued by transgenic expression of wild-typenosmRNA but not by a localization-compromisednosderivative. While localization is essential fornosfunction in dendrite morphogenesis, the mechanism underlying the transport ofnosRNP particles was unknown. We investigated the mechanism of dendriticnosmRNA localization by analyzing requirements fornosRNP particle motility in class IV da neuron dendrites through live imaging of fluorescently labelednosmRNA. We show that dynein motor machinery components mediate transport ofnosmRNA in proximal dendrites. Two factors, the RNA-binding protein Rumpelstiltskin and the germ plasm protein Oskar, which are required for diffusion/entrapment-mediated localization ofnosduring oogenesis, also function in da neurons for formation and transport ofnosRNP particles. Additionally, we show thatnosregulates neuronal function, most likely independent of its dendritic localization and function in morphogenesis. Our results reveal adaptability of localization factors for regulation of a target transcript in different cellular contexts.

The ubiquitous mRNA binding protein human antigen R (HuR), a member of the embryonal lethal abnormal vision (ELAV) protein family has a critical impact on the posttranscriptional control of AU-rich element (ARE) bearing mRNA regulons implied in inflammation, senescence and carcinogenesis. HuR in addition to mRNA stability can affect many other aspects of mRNA processing including splicing, polyadenylation, translation, modulation of miRNA repression and intracellular mRNA trafficking. Since many of the identified HuR mRNA targets (“HuR mRNA regulons”) encode tumor-related proteins, HuR is not only discussed as an useful biomarker but also as promising therapeutic target for treatment of various human cancers. HuR which is most abundantly localized in the nucleus is translocated to the cytoplasm which is fundamental for most of the described HuR functions on target mRNAs. Accordingly, an elevation in cytoplasmic HuR was found in many tumors and correlated with a high grade of malignancy and a poor prognosis of patients. Therefore, direct interference with the intracellular trafficking of HuR offers an attractive approach to intervene with pathologically deregulated HuR functions. Data from several laboratories implicate that the integrity of the cytoskeleton is critical for HuR-mediated intracellular mRNA localization and translation. This review will particularly focus on drugs which have proven a direct inhibitory effect on HuR translocation. Based on the results from those studies, we will also discuss on the principle value of targeting cytoskelton-dependent transport of HuR by natural or synthetic inhibitors as a potential therapeutic avenue for interfering with dysregulated posttranscriptional HuR mRNA regulons and related tumor cell functions. In spite of that, interfering with cytoplasmic HuR transport could highlight a so far underestimated action of microtubule inhibitors clinically used for cancer chemotherapy.

Intracellular mRNA localization and translation are ways to achieve asymmetric protein sorting in polarized cells, and they play fundamental roles in cell-fate decisions and body patterning during animal development. These processes are regulated by the interplay between cis-acting elements and trans-acting RNA-binding proteins that form and occur within a ribonucleoprotein (RNP) complex. Recent studies in the Drosophila oocyte have revealed that RNP complex assembly in the nucleus is critical for the regulation of cytoplasmic mRNA localization and translation. Furthermore, several trans-acting factors promote the reorganization of target mRNAs in the cytoplasm into higher-order RNP granules, which are often visible by light microscopy. Therefore, RNA localization and translation are likely to be coupled within these RNP granules. Notably, diverse cytoplasmic RNP granules observed in different cell types share conserved sets of proteins, suggesting they have fundamental and common cellular functions.

Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of proteins. Previous studies have revealed that in a number of cell types, different mRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into separate or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, we analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 different mRNAs to the bud with the help of the Myo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the RNA tag sequences. Using three-dimensional, single-particle tracking with dual-color detection, we have tracked the transport of two different localized mRNA species in real time. Our observations show that different localized mRNAs are coassembled into common RNP particles and cotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form separate particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.

Intracellular mRNA localization directs protein synthesis to particular subcellular domains to establish embryonic polarity in a wide range of vertebrates and invertebrates. In Drosophila, bicoid (bcd) mRNA is pre-localized at the anterior of the oocyte. After fertilization, this RNA is translated to produce a Bcd protein gradient that determines anterior cell fates [1, 2]. Recent analysis of bcd mRNA during late stages of oogenesis led to a model for steady-state localization of bcd by continual active transport [3]. Here, we elucidate the path and mechanism of sustained bcd mRNA transport by direct observation of bcd RNA particle translocation in living oocytes. However, this mechanism cannot explain maintenance of bcd localization throughout the end of oogenesis, when microtubules disassemble in preparation for embryogenesis [4, 5] or retention of bcd at the anterior in mature oocytes, which can remain dormant, but developmentally competent, for weeks prior to fertilization [6]. Through temporal analysis of bcd RNA particle dynamics, we show that bcd mRNA shifts from continuous active transport to stable actin-dependent anchoring at the end of oogenesis, ensuring the developmental integrity of the oocyte during dormancy. Egg activation triggers release of bcd from the anterior cortex for proper deployment in the fertilized egg, probably through reorganization of the actin cytoskeleton. These findings uncover a surprising parallel between flies and frogs, as cortically tethered Xenopus Vg1mRNA undergoes a similar redistribution during oocyte maturation [7]. Our results highlight a conserved mechanism used by invertebrates and vertebrates to regulate mRNA anchoring and redeployment during the oocyte-to-embryo transition.

Messenger RNA (mRNA) localization plays an important role in various cellular functions. To date, two general mechanisms have been identified for intracellular mRNA localization. The first one was identified by Blobel and colleagues more than three decades ago, by which mRNAs encoding for membrane and secreted proteins are targeted to the endoplasmic reticulum (ER) in a signal peptide dependent manner.1 The second mechanism is for the intracellular targeting of mRNAs encoding cytosolic proteins, which is dependent on specific sequence on the mRNA called zipcode.2 Recently, we have identified a new mechanism which targets Dia1 mRNA to the perinuclear ER in a zipcode-independent manner, even though the mRNA encodes a cytosolic protein.3 Here, we provide an updated discussion on how the Dia1 mRNA is targeted and what might be its physiological significance.

We previously described the cDNA cloning and expression patterns of actin genes from amphioxus Branchiostoma floridae (Kusakabe, R., Kusakabe, T., Satoh, N., Holland, N.D., Holland, L.Z., 1997. Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development. Dev. Genes Evol. 207, 203-215). In the present paper, we report the characterization of cDNA clones for actin genes from a closely related species, Branchiostoma belcheri, and the exon-intron organization of B. floridae actin genes. Each of these two amphioxus species has two types of actin genes, muscle and cytoplasmic. The coding and non-coding regions of each type are well-conserved between the two species. A comparison of nucleotide sequences of muscle actin genes between the two species suggests that a gene conversion may have occurred between two B. floridae muscle actin genes BfMA1 and BfMA2. From the conserved positions of introns between actin genes of amphioxus and those of other deuterostomes, the evolution of deuterostome actin genes can be inferred. Thus, the presence of an intron at codon 328/329 in vertebrate muscle and cytoplasmic actin genes but not in any known actin gene in other deuterostomes suggests that a gene conversion may have occurred between muscle and cytoplasmic actin genes during the early evolution of the vertebrates after separation from other deuterostomes. A Southern blot analysis of genomic DNA revealed that the amphioxus genome contains multiple muscle and cytoplasmic actin genes. Some of these actin genes seem to have arisen from recent duplication and gene conversion. Our findings suggest that the multiple genes encoding muscle and cytoplasmic actin isoforms arose independently in each of the three chordate lineages, and gene duplications and gene conversions established the extant actin multigene family during the evolution of chordates.

The cephalochordate amphioxus is thought to share a common ancestor with vertebrates. To investigate the evolution of developmental mechanisms in chordates, cDNA clones for two amphioxus actin genes, BfCA1 and BfMA1, were isolated. BfCA1 encodes a cytoplasmic actin and is expressed in a variety of tissues during embryogenesis, beginning in the dorsolateral mesendoderm of the mid-gastrula. At the open neural plate stage, BfCA1 transcripts accumulate at the bases of the neuroectodermal cells adjacent the presumptive notochord. The 3' untranslated region of BfCA1 contains a sequence that is similar to the "zipcode" sequence of chicken β-cytoplasmic actin gene, which is thought to direct intracellular mRNA localization. BfCA1 is also expressed in the notochord through the early larval stage, in the pharynx and in the somites at the onset of muscle-cell differentiation. BfMA1 is a vertebrate-type muscle actin gene, although the deduced amino acid sequence is fairly divergent. Transcripts first appear in the early neurula in the somites as they begin to differentiate into axial muscle cells and persist into the adult stage. In young adults, transcripts are localized in the Z-discs of the muscle cells. Smooth muscle cells around the gill slits and striated muscle cells in the pterygeal muscle also express BfMA1; however, there is never any detectable expression in the notochord, which is a modified striated muscle. Together with the alkali myosin light chain gene AmphiMLC-alk, the sequence and muscle-specific expression of BfMA1 implies a conserved mechanism of muscle cell differentiation between amphioxus and vertebrates. Evolution of the chordate actin gene family is discussed based on molecular phylogenetic analysis and expression patterns of amphioxus actin genes.

Signal-peptide-mediated ER localization of mRNAs encoding for membrane and secreted proteins, and RNA-zipcode-mediated intracellular targeting of mRNAs encoding for cytosolic proteins are two well-known mechanisms for mRNA localization. Here, we report a previously unidentified mechanism by which mRNA encoding for Dia1, a cytosolic protein without the signal peptide, is localized to the perinuclear ER in an RNA-zipcode-independent manner in fibroblasts. Dia1 mRNA localization is also independent of the actin and microtubule cytoskeleton but requires translation and the association of Dia1 nascent peptide with the ribosome–mRNA complex. Sequence mapping suggests that interactions of the GTPase binding domain of Dia1 peptide with active Rho are important for Dia1 mRNA localization. This mechanism can override the β-actin RNA zipcode and redirect β-actin mRNA to the perinuclear region, providing a new way to manipulate intracellular mRNA localization.

The intracellular localization of mRNA is an important mechanism for targeting proteins to their sites of function. Localized transcripts play key roles ranging from body axis determination to synapse plasticity, and recent studies highlight how common localized transcripts are in a variety of model organisms. In this review, we consider the full range of known mechanisms for delivering transcripts and anchoring them at their site of function. Finally, we evaluate the progress that has been made in the nervous system in understanding the mechanism and function of mRNA localization and translation regulation.

Two neuropeptide precursor cDNAs (LUQ-1 and L5-67) have been recently isolated from the Left Upper Quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica (Shyamala, Fisher, and Scheller, 1986; Wickham and DesGroseillers, 1991). Using in situ hybridization techniques as well as dot blot and polymerase chain reaction (PCR) assays, we have studied the expression of these genes in the central nervous system (CNS) of Aplysia californica. The LUQ-1 gene was found to be expressed in neuron L5 in the abdominal ganglion, whereas the expression of the L5-67 gene was observed in the other four LUQ cells (L2-4 and L6). When in situ hybridization was performed on paraffin sections of the abdominal ganglion, clusters of smaller cells located in the left hemiganglion, were also found to express either the LUQ-1 or the L5-67 gene, never both. In many sections, the mRNAs coding for the two neuropeptides were found not only in cell bodies but also in the axon of individual LUQ neurons and even as far as the pericardial nerve. The presence of neuropeptide mRNA in axons, pericardial nerve, and kidney has been confirmed by polymerase chain reaction. A specific, although diffuse hybridization in the left upper quadrant also suggests that mRNA is present in the neuritic field. Taken together these results indicate that neuron L5 is the only giant neuron expressing the LUQ-1 gene and might therefore have a physiological function different from the other four LUQ cells. Neuropeptide mRNAs were also found in the axon and/or the neuritic field of giant neurons and could play important roles related to cell signalling in axons and nerve termini.

Intracellular mRNA localization is a common mechanism of post-transcriptional regulation of gene expression. In a wide range of organisms, mRNA localization coupled with translational regulation target the proteins to their site of function. Here, we describe recent exciting evidence that some mRNAs are transported as particles along the cytoskeleton by the molecular motors dynein, kinesin or myosin. We discuss the key questions of how localized mRNAs might be linked to motors and what determines their cytoplasmic destinations.

Intracellular mRNA localization plays an important role in post transcriptional gene regulation by targeting protein synthesis to a specific cellular compartment. mRNA localization has been extensively investigated with fluorescence in situ hybridization and live cell imaging in various model organisms. However, quantitative measurement of the translation of localizing mRNA with high spatiotemporal precision remains challenging. β-actin mRNA has been shown to localize to the leading edge of primary fibroblasts and provides an excellent model system to address this problem. The localization involves a cis-acting zipcode sequence and a trans-acting zipcode binding protein (ZBP1). Here we employ fluorescence fluctuation spectroscopy (FFS) to investigate the localization of β-actin mRNA and its association with ribosome subunits in live cell. Two technical advances make this experiment possible. First, we have developed a mouse model in which the β-actin gene has 24XMS2 sequences inserted. Furthermore, this mouse has been crossed with a ZBP1 knock out mouse. We generate primary mouse embryonic fibroblasts from these mice. By expressing GFP-MS2 capsid protein in the cell, all endogenous β-actin mRNA are fluorescently labeled. In addition, mCherry labeled ZBP1 or ribosome subunit are introduced into the cells to visualize their interaction with the β-actin mRNA by FFS. Second, we have developed a dual-color brightness analysis tool, specifically hetero-species partition analysis, to measure the interaction between the GFP-MS2 labeled mRNA and another mCherry labeled factor. We systematically measured the interaction between the β-actin mRNA and the ZBP1/ribosome in the perinuclear region and leading edge. Our data shows that β-actin mRNA binds more ZBP1 but less ribosome in the perinuclear region than in the leading edge, which supports the model that ZBP1 represses β-actin mRNA association with ribosome in the nuclear periphery until it reaches the leading edge.

Intracellular mRNA localization is an effective mechanism for protein targeting leading to functional polarization of the cell. The mechanisms controlling mRNA localization and specifically how the actin and microtubule (MT) cytoskeletons cooperate in this process are not well understood. In Drosophila, Oskar protein accumulation at the posterior pole of the oocyte is required for embryonic development 1, 2, 3 and 4 and is achieved by the transport of oskar mRNA and its exclusive translation at the posterior pole 1, 5, 6 and 7. oskar mRNA localization requires the activity of the MT-based motor Kinesin [8], as well as the formation of a transport-competent ribonucleoprotein (RNP) complex [9]. Here, we show that didum, encoding the Drosophila actin-based motor Myosin-V 10 and 11, is a new posterior group gene that promotes posterior accumulation of Oskar. Myosin-V associates with the oskar mRNA transport complex preferentially at the oocyte cortex, revealing a short-range actomyosin-based mechanism that mediates the local entrapment of oskar at the posterior pole. Our results also show that Myosin-V interacts with Kinesin heavy chain and counterbalances Kinesin function, preventing ectopic accumulation of oskar in the cytoplasm. Our findings reveal that a balance of microtubule- and actin-based motor activities regulates oskar mRNA localization in the Drosophila oocyte.

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. © 2010 Elsevier Ltd.

A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.