Daniel St Johnston

staufen, a gene required to localize maternal RNAs in the Drosophila egg

The posterior group gene staufen is required both for the localization of maternal determinants to the posterior pole of the Drosophila egg and for bicoid RNA to localize correctly to the anterior pole. We report the cloning and sequencing of staufen and show that staufen protein is one of the first molecules to localize to the posterior pole of the oocyte, perhaps in association with oskar RNA. Once localized, staufen is found in the polar granules and is required to hold other polar granule components at the posterior pole. By the time the egg is laid, staufen protein is also concentrated at the anterior pole, in the same region as bicoid RNA.

The intracellular localization of messenger RNAs

As I hope this review has made clear, mRNA localization plays an important role in directing specific proteins to their correct position within a cell. Although the study of this process is still in its infancy, it is already apparent that there are several ways that mRNAs can be targeted to particular subcellular sites. However, the molecular mechanisms responsible for these different localization pathways are still largely obscure, and their elucidation must await the identification of the specific factors that mediate the interactions between the localized mRNAs and more general components such as the cytoskeleton. Most examples of localized mRNAs are likely to share several common features. First, the site of localization will be determined by the preexisting polarity of the cell, and this will most often depend on the organization of the cytoskeleton, either directly, in the case of active transport, or indirectly, when localization is mediated by localized anchoring sites or stability factors. Second, mRNA localization is likely to be tightly coupled to translational control. If it is important for a cell to synthesize a protein in a particular place, then the translation of the mRNA must be repressed until it is localized. Indeed, there are already several examples where the direct linkage between translational control and localization has been demonstrated, and these are discussed in the accompanying review by Curtis et al. (1995).

Moving messages: the intracellular localization of mRNAs.

mRNA localization is a common mechanism for targeting proteins to regions of the cell where they are required. It has an essential role in localizing cytoplasmic determinants, controlling the direction of protein secretion and allowing the local control of protein synthesis in neurons. New methods for in vivo labelling have revealed that several mRNAs are transported by motor proteins, but how most mRNAs are coupled to these proteins remains obscure.Key PointsmRNA localization is a widespread mechanism for targeting proteins to the regions of a cell where they are required, and has an important role in localizing cytoplasmic determinants, targeting protein secretion, and synaptic plasticity.mRNAs can be localized by four different mechanisms: local synthesis, local protection from degradation, diffusion and anchoring, or active transport by molecular motors. The latter seems to be the most common mechanism, although it is also the most difficult one to show.The only case in which it is known how an mRNA is linked to a motor is ASH1 mRNA in yeast. ASH1 mRNA is linked through She3 and She2 to the myosin, Myo4, which then transports the mRNA along actin cables into the bud tip. β-Actin mRNA might be localized by a similar mechanism in chicken fibroblasts, whereas prospero mRNA is localized to the basal cortex of Drosophila melanogaster neuroblasts by apical exclusion through myosin II and basal anchoring by a myosin VI.Dynein transports pair-rule transcripts to the apical side of the D. melanogaster syncytial blastoderm embryo, in a process that requires the BicD and EGL proteins. This pathway also mediates nurse-cell-to-oocyte transport and apical mRNA localization in neuroblasts. bicoid and gurken mRNAs are also believed to be localized by dynein in the D. melanogaster oocyte, but must discriminate between different populations of microtubules to localize to the anterior and dorsal–anterior cortex, respectively.Kinesin is required for the posterior localization of oskar mRNA in the D. melanogaster oocyte, but it remains to be shown whether it actively transports the mRNA there. Several other mRNAs might also be transported by kinesin, such as MBP mRNA in mammalian oligodendrocytes, Vg1 mRNA in the Xenopus laevis oocyte and CaMKIIα mRNA in mammalian dendrites, and the latter co-localizes with several RNA-binding proteins that form a complex with the kinesin tail.Cis-acting localization elements usually reside in 3′ untranslated regions, but are occasionally found elsewhere in the mRNA. They are sometimes absent from the mature message, as is the case for oskar mRNA, where splicing of the first intron is necessary for transport to the posterior of the oocyte. The simplest localization element is the 10-nucleotide A2RE in MBP mRNA, which binds hnRNPA2 to direct the localization into oligodendrocyte processes. All other localization elements seem to be more complex and can contain multiple redundant signals, or form intricate secondary or higher-order structures.It is now becoming apparent that the localization of many mRNAs requires the stepwise assembly of large RNA-protein complexes, in which some proteins associate with the mRNA in the nucleus, and others in the cytoplasm. Several RNA–binding proteins have been implicated in the localization of various mRNAs, such as Staufen, ZBP1/VERA and hnRNPA/B family members.AbstractmRNA localization is a common mechanism for targeting proteins to regions of the cell where they are required. It has an essential role in localizing cytoplasmic determinants, controlling the direction of protein secretion and allowing the local control of protein synthesis in neurons. New methods for in vivo labelling have revealed that several mRNAs are transported by motor proteins, but how most mRNAs are coupled to these proteins remains obscure.

Seeing Is Believing: The Bicoid Morphogen Gradient Matures

Although Cell has a long history of publishing some of the most significant advances in developmental biology, the back to back papers by Driever and Nüsslein-Volhard on the role of the Bicoid gradient in patterning the Drosophila embryo stand out as the first molecular demonstration of two of the longest standing concepts of the field, namely localized cytoplasmic determinants and morphogen gradients. Here we discuss the impact of this ground-breaking work and review recent results on bicoid mRNA localization and the dual role of Bicoid as a transcription and translation factor.

The art and design of genetic screens: Drosophila melanogaster

Key Points The success of Drosophila melanogaster as a model organism is largely due to the power of forward genetic screens to identify the genes that are involved in a biological process. Although traditional genetic screens, such as zygotic lethal screens, have been useful for identifying the genes that act early in fly development, more sophisticated and versatile screens have been developed. Modifier screens, in which genes are identified by their ability to alter the phenotype of flies that are genetically sensitized for the process of interest, are useful for finding the components of signal-transduction pathways. Clonal screens, in which cells that are homozygous for a mutation of interest can be made in an otherwise heterozygous animal through targeted mitotic recombination (using the Flp/FRT system), allow researchers to identify genes that act in a specific tissue at any stage of development. A modification of this technique, in which clones are made in the germ line, helps to find genes that are maternally supplied to the embryo. Mutant screens do not always identify all of the genes for which loss-of-function mutations give the phenotype of interest, as with genes that have redundant functions. In these cases, the GAL4–UAS system can be used to screen for mis- or overexpression phenotypes. The success of a genetic screen depends in large part on its design. As mapping and identifying genes that result from a screen can be very laborious, it is often preferable to sacrifice speed and the number of genes recovered in favour of a phenotype that is directly related to the process of interest. Genetic screens will continue to be useful in years to come, even once all genes have been identified. The functional characterization of genes relies on having allelic variants of each gene, which, for the time being, only genetic screens can provide.AbstractThe success of Drosophila melanogaster as a model organism is largely due to the power of forward genetic screens to identify the genes that are involved in a biological process. Traditional screens, such as the Nobel-prize-winning screen for embryonic-patterning mutants, can only identify the earliest phenotype of a mutation. This review describes the ingenious approaches that have been devised to circumvent this problem: modifier screens, for example, have been invaluable for elucidating signal-transduction pathways, whereas clonal screens now make it possible to screen for almost any phenotype in any cell at any stage of development.The success of Drosophila melanogaster as a model organism is largely due to the power of forward genetic screens to identify the genes that are involved in a biological process. Traditional screens, such as the Nobel-prize-winning screen for embryonic-patterning mutants, can only identify the earliest phenotype of a mutation. This review describes the ingenious approaches that have been devised to circumvent this problem: modifier screens, for example, have been invaluable for elucidating signal-transduction pathways, whereas clonal screens now make it possible to screen for almost any phenotype in any cell at any stage of development.

Cell Polarity in Eggs and Epithelia: Parallels and Diversity

Cell polarity, the generation of cellular asymmetries, is necessary for diverse processes in animal cells, such as cell migration, asymmetric cell division, epithelial barrier function, and morphogenesis. Common mechanisms generate and transduce cell polarity in different cells, but cell type-specific processes are equally important. In this review, we highlight the similarities and differences between the polarity mechanisms in eggs and epithelia. We also highlight the prospects for future studies on how cortical polarity interfaces with other cellular processes, such as morphogenesis, exocytosis, and lipid signaling, and how defects in polarity contribute to tumor formation.

Drosophila PAR-1 and 14-3-3 Inhibit Bazooka/PAR-3 to Establish Complementary Cortical Domains in Polarized Cells

PAR-1 kinases are required for polarity in diverse cell types, such as epithelial cells, where they localize laterally. PAR-1 activity is believed to be transduced by binding of 14-3-3 proteins to its phosphorylated substrates, but the relevant targets are unknown. We show that PAR-1 phosphorylates Bazooka/PAR-3 on two conserved serines to generate 14-3-3 binding sites. This inhibits formation of the Bazooka/PAR-6/aPKC complex by blocking Bazooka oligomerization and binding to aPKC. In epithelia, this complex localizes apically and defines the apical membrane, whereas Bazooka lacking PAR-1 phosphorylation/14-3-3 binding sites forms ectopic lateral complexes. Lateral exclusion by PAR-1/14-3-3 cooperates with apical anchoring by Crumbs/Stardust to restrict Bazooka localization, and loss of both pathways disrupts epithelial polarity. PAR-1 also excludes Bazooka from the posterior of the oocyte, and disruption of this regulation causes anterior-posterior polarity defects. Thus, antagonism of Bazooka by PAR-1/14-3-3 may represent a general mechanism for establishing complementary cortical domains in polarized cells.

An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay

The specification of both the germ line and abdomen in Drosophila depends on the localization of oskar messenger RNA to the posterior of the oocyte. This localization requires several trans-acting factors, including Barentsz and the Mago–Y14 heterodimer, which assemble with oskar mRNA into ribonucleoprotein particles (RNPs) and localize with it at the posterior pole. Although Barentsz localization in the germ line depends on Mago–Y14, no direct interaction between these proteins has been detected. Here, we demonstrate that the translation initiation factor eIF4AIII interacts with Barentsz and is a component of the oskar messenger RNP localization complex. Moreover, eIF4AIII interacts with Mago–Y14 and thus provides a molecular link between Barentsz and the heterodimer. The mammalian Mago (also known as Magoh)–Y14 heterodimer is a component of the exon junction complex. The exon junction complex is deposited on spliced mRNAs and functions in nonsense-mediated mRNA decay (NMD), a surveillance mechanism that degrades mRNAs with premature translation-termination codons. We show that both Barentsz and eIF4AIII are essential for NMD in human cells. Thus, we have identified eIF4AIII and Barentsz as components of a conserved protein complex that is essential for mRNA localization in flies and NMD in mammals.

RNA recognition by a Staufen double-stranded RNA-binding domain

The double‐stranded RNA‐binding domain (dsRBD) is a common RNA‐binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem–loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA‐binding activity of dsRBD3 is required in vivo for Staufen‐dependent localization of bicoid and oskar mRNAs. Using high‐resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem–loop. The dsRBD recognizes the shape of A‐form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix α1 interacts with the single‐stranded loop that caps the RNA helix. Interactions between helix α1 and single‐stranded RNA may be important determinants of the specificity of dsRBD proteins.

Activity-dependent decrease of excitability in rat hippocampal neurons through increases in Ih

Hippocampal long-term potentiation (LTP) induced by theta-burst pairing of Schaffer collateral inputs and postsynaptic firing is associated with localized increases in synaptic strength and dendritic excitability. Using the same protocol, we now demonstrate a decrease in cellular excitability that was blocked by the h-channel blocker ZD7288. This decrease was also induced by postsynaptic theta-burst firing alone, yet it was blocked by NMDA receptor antagonists, postsynaptic Ca2+ chelation, low concentrations of tetrodotoxin, ω-conotoxin MVIIC, calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors and a protein synthesis inhibitor. Increasing network activity with high extracellular K+ caused a similar reduction of cellular excitability and an increase in h-channel HCN1 protein. We propose that backpropagating action potentials open glutamate-bound NMDA receptors, resulting in an increase in Ih and a decrease in overall excitability. The occurrence of such a reduction in cellular excitability in parallel with synaptic potentiation would be a negative feedback mechanism to normalize neuronal output firing and thus promote network stability.