Reversible inhibition of translation by Xenopus oocyte-specific proteins

A characteristic of growing oocytes of all animal species is the synthesis and accumulation of messenger RNA which is destined to be used primarily by the early embryo. The mechanism(s) which regulates the translation of this maternal mRNA remains unknown. However, the inability of the oocyte to translate all of its putative mRNA has been attributed to at least three limitations: (1) The rate of translation is limited by the availability of components of the translational apparatus other than mRNA, (2) the structural organization of the mRNA prevents translation, and (3) proteins associated with the mRNA prevent translation. Several investigators have suggested that proteins associated with maternal mRNA suppress translation in sea urchin eggs, although others claim that such results may be due to experimental artefacts. Oocyte-specific proteins have been identified in association with non-translating poly(A)+ mRNAs from Xenopus laevis oocytes, and we report here that when these proteins are reconstituted with mRNAs in vitro the translation of the mRNAs in vitro is reversibly repressed. The implication is that these proteins are involved in the regulation of translation of stored maternal mRNAs.     

Drosophila nurse cells produce a posterior signal required for embryonic segmentation and polarity

The segmental pattern of insect embryos depends on influences from morphogenetic centres near each of the egg poles. In Drosophila, maternal effect mutations are known that impair the normal function of each centre. Injection of wild-type cytoplasm into mutant eggs has revealed that morphogenetic signals localized at the anterior and posterior pole of eggs can be transplanted. We show here that these activities can also be detected during oogenesis. Posterior activity can be recovered at an early stage (stage 10, ref. 5) from the oocyte-nurse cell complex, but anterior activity can only be detected in the mature oocytes (stage 14). We conclude that the bicoid-dependent anterior signal, although produced by the nurse cells, does not become active before it is localized to the anterior egg pole, whereas posterior activity can be detected in the nurse cells before, and therefore independently of, its localization to the posterior egg pole.     

The Xenopus localized messenger RNA An3 may encode an ATP-dependent RNA helicase

The maternal messenger RNA An3 was originally identified localized to the animal hemisphere of Xenopus laevis oocytes, eggs and early embryos. Xenopus embryos depend on mRNA and protein present in the egg before fertilization (maternal molecules) to provide the information needed for early development. Localization of maternal mRNA gives cells derived from different regions of the egg distinctive capacities for protein synthesis. We show here that An3 mRNA encodes a protein with 74% identity to a protein encoded by the testes-specific mRNA PL10 found in mouse, which is proposed to have RNA helicase activity. Because the gene encoding An3 mRNA is reactivated after gastrulation and remains active throughout embryogenesis, we have examined its distribution in embryonic and adult tissues. Unlike PL10 mRNA, which is primarily restricted to the testes, An3 mRNA is broadly distributed in later development.     

cDNA cloning of bovine substance-K receptor through oocyte expression system

The neuropeptide receptors which are present in very small quantities in the cell and are embedded tightly in the plasma membrane have not been well characterized. Mammals contain three distinct tachykinin neuropeptides, substance P, substance K and neuromedin K, and it has been suggested that there are multiple tachykinin receptors. By electrophysiological measurement, we have previously shown that Xenopus oocytes injected with brain and stomach mRNAs faithfully express mammalian substance-P and substance-K receptors, respectively. Here we report the isolation of the cDNA clone for bovine substance-K receptor (SKR) by extending this method to develop a new cloning strategy. We constructed a stomach cDNA library with a cloning vector that allowed in vitro synthesis of mRNAs and then identified a particular cDNA clone by testing for receptor expression following injection of the mRNAs synthesized in vitro into the oocyte system. Because oocytes injected with exogenous mRNAs can express numerous receptors and channels, our new strategy will be applicable in the general molecular cloning of these proteins. The result provides the first indication that the neuropeptide receptor has sequence similarity with rhodopsin-type receptors (the G-protein-coupled receptor family) and thus possesses multiple membrane-spanning domains.     

macho-1 encodes a localized mRNA in ascidian eggs that specifies muscle fate during embryogenesis

Maternal information stored in particular regions of the egg cytoplasm has an important function in the determination of developmental fate during early animal development. Ascidians show mosaic development; such autonomous development has been taken as evidence that prelocalized ooplasmic factors specify tissue precursor cells during embryogenesis. Interest has been concentrated on the mechanisms underlying the formation of muscle cells in the tail, as yellow-coloured myoplasm in eggs is preferentially segregated into muscle-lineage blastomeres. Here we show that maternal messenger RNA of the macho-1 gene is a determinant of muscle fate in the ascidian Halocynthia roretzi. The macho-1 mRNA encodes a zinc-finger protein, and the mRNA is localized to the myoplasm of eggs. Depletion of the mRNA specifically resulted in the loss of primary muscle cells in the tail, as shown by the expression of muscle-specific molecular markers. The myoplasm of macho-1-deficient eggs lost its ability to promote muscle formation. Injection of synthesized macho-1 mRNA caused ectopic muscle formation in non-muscle-lineage cells. Our results indicate that macho-1 maybe both required and sufficient for specification of muscle fate, and that the mRNA is a genuine, localized muscle determinant.     

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.     

Internal initiation of translation mediated by the 5' leader of a cellular mRNA.

A Robosome-scanning model has been proposed to explain the initiation of eukaryotic messenger RNAs in which binding of the 43S ternary ribosomal subunit near or at the 5' end of the mRNA is facilitated by an interaction between the methylated cap-structure at the end of the mRNA and the cap-binding protein complex eIF-4F. But picornaviral mRNAs do not have a 5' terminal cap structure and are translated by internal ribosome binding. A cellular mRNA, encoding the immunoglobulin heavy-chain binding protein, can be translated in poliovirus-infected cells at a time when cap-dependent translation of host cell mRNAs is inhibited. We report here that the 5' leader of the binding protein mRNA can directly confer internal ribosome binding to an mRNA in mammalian cells, indicating that translation initiation by an internal ribosome-binding mechanism is used by eukaryotic mRNAs.     

TOP mRNA及其5＇TOP序列的基因表达调控作用

TOP mRNA是以胞嘧啶为起始,紧接5～15个聚嘧啶序列的一类mRNA。它在脊椎动物细胞mRNA中占很大比重,是一类与编码核糖体蛋白、翻译延伸因子和起始因子等翻译元件相关的mRNA,其5＇TOP序列对细胞生长、分化、发育具有重要作用。目前的研究发现此类mRNA的表达受到mTOR信号转导路径的调控。并且它们可能通过与La或CNBP等反式作用因子的作用调控基因表达。     

Localized maternal orthodenticle patterns anterior and posterior in the long germ wasp Nasonia

The Bicoid (Bcd) gradient in Drosophila has long been a model for the action of a morphogen in establishing embryonic polarity. However, it is now clear that bcd is a unique feature of higher Diptera. An evolutionarily ancient gene, orthodenticle (otd), has a bcd-like role in the beetle Tribolium. Unlike the Bcd gradient, which arises by diffusion of protein from an anteriorly localized messenger RNA, the Tribolium Otd gradient forms by translational repression of otd mRNA by a posteriorly localized factor. These differences in gradient formation are correlated with differences in modes of embryonic patterning. Drosophila uses long germ embryogenesis, where the embryo derives from the entire anterior-posterior axis, and all segments are patterned at the blastoderm stage, before gastrulation. In contrast, Tribolium undergoes short germ embryogenesis: the embryo arises from cells in the posterior of the egg, and only anterior segments are patterned at the blastoderm stage, with the remaining segments arising after gastrulation from a growth zone. Here we describe the role of otd in the long germband embryo of the wasp Nasonia vitripennis. We show that Nasonia otd maternal mRNA is localized at both poles of the embryo, and resulting protein gradients pattern both poles. Thus, localized Nasonia otd has two major roles that allow long germ development. It activates anterior targets at the anterior of the egg in a manner reminiscent of the Bcd gradient, and it is required for pre-gastrulation expression of posterior gap genes.     

Origin of a gene regulatory mechanism in the evolution of echinoderms

A rich diversity of ancient sea urchin lineages survives to the present. These include several advanced orders as well as the cidaroids, which represent the group ancestral to all other sea urchins. Here we show that all advanced groups of sea urchins examined possess in their eggs a class of maternal messenger RNA (mRNA) encoded by the evolutionarily highly conserved alpha-subtype histone genes. The maternal histone mRNAs are unique in their time of accumulation in oogenesis, their localization in the egg nucleus and their delayed timing of translation after fertilization. Cidaroid sea urchins as well as other echinoderm classes, such as starfish and sea cucumbers, possess the genes but do not have maternal alpha-subtype histone mRNAs in their eggs. Thus, although all the echinoderms examined transcribe alpha-subtype histone genes during embryogenesis, the expression of these genes as maternal mRNAs is confined to advanced sea urchins. The fossil record allows us to pinpoint the evolution of this mode of expression of alpha-histone genes to the time of the splitting of advanced sea urchin lineages from the ancestral cidaroids in a radiation which occurred in a relatively brief interval of time approximately 190-200 Myr ago. The origin of a unique gene regulatory mechanism can thus be correlated with a set of macroevolutionary events.     

An efflux transporter of silicon in rice

Silicon is an important nutrient for the optimal growth and sustainable production of rice. Rice accumulates up to 10% silicon in the shoot, and this high accumulation is required to protect the plant from multiple abiotic and biotic stresses. A gene, Lsi1, that encodes a silicon influx transporter has been identified in rice. Here we describe a previously uncharacterized gene, low silicon rice 2 (Lsi2), which has no similarity to Lsi1. This gene is constitutively expressed in the roots. The protein encoded by this gene is localized, like Lsi1, on the plasma membrane of cells in both the exodermis and the endodermis, but in contrast to Lsi1, which is localized on the distal side, Lsi2 is localized on the proximal side of the same cells. Expression of Lsi2 in Xenopus oocytes did not result in influx transport activity for silicon, but preloading of the oocytes with silicon resulted in a release of silicon, indicating that Lsi2 is a silicon efflux transporter. The identification of this silicon transporter revealed a unique mechanism of nutrient transport in plants: having an influx transporter on one side and an efflux transporter on the other side of the cell to permit the effective transcellular transport of the nutrients.     

神经细胞树突中mRNA的运输

将mRNA靶定到神经细胞树突中, 对区域化蛋白质合成和神经功能的发挥起重要作用。神经细胞mRNA存在于包含不同种类调控mRNA 定位、稳定和翻译组分的颗粒中。该颗粒由驱动蛋白1(kinesin 1) 沿微管运输。定向运输产生的mRNA不对称分布是突触可塑性、学习和记忆所必需的。     

Heteromultimeric channels formed by rat brain potassium-channel proteins

An important step towards understanding the molecular basis of the functional diversity of voltage-gated K+ channels in the mammalian brain has been the discovery of a family of genes encoding rat brain K+ channel-forming (RCK) proteins. All species of these RCK proteins form homomultimeric voltage-gated K+ channels with distinct functional characteristics in Xenopus laevis oocytes following injection of the respective cRNAs. RCK-specific mRNAs are coexpressed in several regions of the brain, suggesting that RCK proteins also assemble into heteromultimeric K+ channels. In addition expression experiments with fractionated poly(A)+ mRNA have suggested that heteromultimeric K+ channels may occur in mammalian brain. We report here that heteromultimeric K+ channels composed of two different RCK proteins (RCK1 and RCK4) assemble after cotransfection of HeLa cells with the corresponding cDNAs and after coinjection of the corresponding cRNAs into Xenopus oocytes. The heteromultimeric RCK1, 4 channel mediates a transient potassium outward current, similar to the RCK4 channel but inactivates more slowly, has a larger conductance and is more sensitive to block by dendrotoxin and tetraethylammonium chloride.