Nancy Standart

How Do MicroRNAs Regulate Gene Expression

Several thousand human genes, amounting to about one-third of the whole genome, are potential targets for regulation by the several hundred microRNAs (miRNAs) encoded in the genome. The regulation occurs posttranscriptionally and involves the ~21-nucleotide miRNA interacting with a target site in the mRNA that generally has imperfect complementarity to the miRNA. The target sites are almost invariably in the 3′-untranslated region of the messenger RNA (mRNA), often in multiple copies. Metazoan miRNAs were previously thought to down-regulate protein expression by inhibiting target mRNA translation at some stage after the translation initiation step, without much effect on mRNA abundance. However, recent studies have questioned these suppositions. With some targets, an increase in the rate of mRNA degradation by the normal decay pathway contributes to the decrease in protein expression. miRNAs can also inhibit translation initiation, specifically the function of the cap-binding initiation factor, eIF4E. Repressed target mRNAs as well as miRNAs themselves accumulate in cytoplasmic foci known as P-bodies, where many enzymes involved in mRNA degradation are concentrated. However, P-bodies may also serve as repositories for the temporary and reversible storage of untranslated mRNA, and reducing the expression (knockdown) of several distinct P-body protein components can alleviate miRNA-mediated repression of gene expression. The human genome encodes several hundred short (~21 residue) microRNAs (miRNAs), which are predicted to collectively regulate several thousand distinct genes, amounting to about one-third of all human genes. MicroRNAs generally interact with their target mRNAs through imperfect or incomplete complementary base-pairing to sites in the 3′-untranslated region of the messenger RNA (mRNA), which are usually present in multiple copies. These interactions result in a decrease in synthesis of the protein encoded by the mRNA, but until very recently, little was known about the mechanism(s) of this repression. An miRNA can decrease the intracellular concentration of its target mRNAs by accelerating the normal process of mRNA degradation and can inhibit the translation or decoding of the target mRNA, with the relative importance of these two mechanisms differing between different miRNA-mRNA pairs. The mechanism of inhibition of translation remains controversial, with some data pointing to inhibition of the translation initiation step, and other results indicative of inhibition at some later stage during the actual decoding of the mRNA sequence. This article reviews these recent data, discusses the controversies that remain unresolved, and makes suggestions for future research.

Asymmetrical β-actin mRNA translation in growth cones mediates attractive turning to netrin-1

Local protein synthesis regulates the turning of growth cones to guidance cues, yet little is known about which proteins are synthesized or how they contribute to directional steering. Here we show that β-actin mRNA resides in Xenopus laevis retinal growth cones where it binds to the RNA-binding protein Vg1RBP. Netrin-1 induces the movement of Vg1RBP granules into filopodia, suggesting that it may direct the localization and translation of mRNAs in growth cones. Indeed, a gradient of netrin-1 activates a translation initiation regulator, eIF-4E-binding protein 1 (4EBP), asymmetrically and triggers a polarized increase in β-actin translation on the near side of the growth cone before growth cone turning. Inhibition of β-actin translation abolishes both the asymmetric rise in β-actin and attractive, but not repulsive, turning. Our data suggest that newly synthesized β-actin, concentrated near sites of signal reception, provides the directional bias for polymerizing actin in the direction of an attractive stimulus.

CPEB Interacts with an Ovary-specific eIF4E and 4E-T in Early Xenopus Oocytes

CPEB (cytoplasmic polyadenylation element-binding protein) is an important regulator of translation in oocytes and neurons. Although previous studies of CPEB in late Xenopus oocytes involve the eIF4E-binding protein maskin as the key factor for the repression of maternal mRNA, a second mechanism must exist, since maskin is absent earlier in oogenesis. Using co-immunoprecipitation and gel filtration assays, we show that CPEB specifically interacts, via protein/protein interactions, with the RNA helicase Xp54, the RNA-binding proteins P100(Pat1) and RAP55, the eIF4E-binding protein 4E-T, and an eIF4E protein. Remarkably, these CPEB complex proteins have been characterized, in one or more organism, as P-body, maternal, or neuronal granule components. We do not detect interactions with eIF4E1a, the canonical cap-binding factor, eIF4G, or eIF4A or with proteins expressed late in oogenesis, including maskin, PARN, and 4E-BP1. The eIF4E protein was identified as eIF4E1b, a close homolog of eIF4E1a, whose expression is restricted to oocytes and early embryos. Although eIF4E1b possesses all residues required for cap and eIF4G binding, it binds m7GTP weakly, and in pull-down assays, rather than binding eIF4G, it binds 4E-T, in a manner independent of the consensus eIF4E-binding site, YSKEELL. Wild type and Y-A mutant 4E-T (which binds eIF4E1b but not eIF4E1a), when tethered to a reporter mRNA, represses its translation in a cap-dependent manner, and injection of eIF4E1b antibody accelerates meiotic maturation. Altogether, our data suggest that CPEB, partnered with several highly conserved RNA-binding partners, inhibits protein synthesis in oocytes using a novel pairing of 4E-T and eIF4E1b.

Cyclin synthesis, modification and destruction during meiotic maturation of the starfish oocyte

The pattern of protein synthesis in oocytes of starfish Marthasterias glacialis changes during 1-methyladenine-induced meiotic maturation. One of the newly synthesized proteins, a major 54-kDa polypeptide, was synthesized continuously after activation but was destroyed abruptly just before appearance of the polar bodies at each meiotic division. This protein thus resembles the cyclin proteins identified in cleaving sea urchin and clam embryos. RNA extracted from oocytes before and after maturation encoded virtually identical polypeptides when translated in the reticulocyte lysate. However, there was poor correspondence between the in vitro translation products and the labelling pattern of intact cells. There was no exact in vitro counterpart to the in vivo-labelled cyclin. Instead, a major polypeptide of 52 kDa was seen which appears to be a precursor of the 54-kDa form of cyclin. The 52-kDa polypeptide was identified as cyclin by hybrid arrest of translation. Cyclin mRNA is ot translated to a significant extent before oocyte activation and is present in oocytes as nonadenylated form. It becomes polyadenylated when the oocytes mature. This behavior is also seen in the case of the mRNA for the small subunit of ribonucleotide reductase, another abundant maternal mRNA whose translation is activated at maturation.

The role of cyclin synthesis, modification and destruction in the control of cell division

Summary This paper reviews our current knowledge of the cyclins based on observations of the oocytes and eggs of sea urchins, clams and frogs. Cyclins are proteins found in all eukaryotes whose special property is rapid destruction at specific stages in the cell cycle. The cyclins fall into three families. A-type cyclins have been found in clams, flies and frogs. B-type cyclins have been found in clams, flies, frogs, sea urchins and fission yeast. A more distantly related family of three genes is found in Saccharomyces cerevisiae. B-type cyclins appear to be required for cells to enter mitosis, and their destruction is thought to be necessary for exit from mitosis. We describe evidence in support of these ideas, and describe various conditions under which cyclin destruction is delayed or deranged. We conclude with a discussion of the relationship between the cyclins and maturation- (or M phase-) promoting factor and some ideas on how the cyclins may work.

RNA-Binding Proteins in Early Development

ABSTRACT RNA-binding proteins play a major part in the control of gene expression during early development. At this stage, the majority of regulation occurs at the levels of translation and RNA localization. These processes are, in general, mediated by RNA-binding proteins interacting with specific sequence motifs in the 3′-untranslated regions of their target RNAs. Although initial work concentrated on the analysis of these sequences and their trans-acting factors, we are now beginning to gain an understanding of the mechanisms by which some of these proteins function. In this review, we will describe a number of different families of RNA-binding proteins, grouping them together on the basis of common regulatory strategies, and emphasizing the recurrent themes that occur, both across different species and as a response to different biological problems.

Abundant and dynamically expressed miRNAs, piRNAs, and other small RNAs in the vertebrate Xenopus tropicalis

Small regulatory RNAs have recently emerged as key regulators of eukaryotic gene expression. Here we used high-throughput sequencing to determine small RNA populations in the germline and soma of the African clawed frog Xenopus tropicalis. We identified a number of miRNAs that were expressed in the female germline. miRNA expression profiling revealed that miR-202-5p is an oocyte-enriched miRNA. We identified two novel miRNAs that were expressed in the soma. In addition, we sequenced large numbers of Piwi-associated RNAs (piRNAs) and other endogenous small RNAs, likely representing endogenous siRNAs (endo-siRNAs). Of these, only piRNAs were restricted to the germline, suggesting that endo-siRNAs are an abundant class of small RNAs in the vertebrate soma. In the germline, both endogenous small RNAs and piRNAs mapped to many high copy number loci. Furthermore, endogenous small RNAs mapped to the same specific subsets of repetitive elements in both the soma and the germline, suggesting that these RNAs might act to silence repetitive elements in both compartments. Data presented here suggest a conserved role for miRNAs in the vertebrate germline. Furthermore, this study provides a basis for the functional analysis of small regulatory RNAs in an important vertebrate model system.

Two Piwi proteins, Xiwi and Xili, are expressed in the Xenopus female germline

The Argonaute superfamily is a large family of RNA-binding proteins involved in gene regulation mediated by small noncoding RNA and characterized by the presence of PAZ and PIWI domains. The family consists of two branches, the Ago and the Piwi clade. Piwi proteins bind to 21-30-nucleotide-long Piwi-interacting RNAs (piRNAs), which map primarily to transposons and repeated sequence elements. Piwi/piRNAs are important regulators of gametogenesis and have been proposed to play roles in transposon silencing, DNA methylation, transcriptional silencing, and/or post-transcriptional control of translation and RNA stability. Most reports to date have concentrated on the Piwi family members in the male germline. We have identified four Piwi proteins in Xenopus and demonstrate that two, namely, Xiwi1b and Xili, are expressed in the oocyte and early embryo. Xiwi1 and Xili are predominantly found in small, separate complexes, and we do not detect significant interaction of Piwi proteins with the cap-binding complex. Putative nuclear localization and export signals were identified in Xiwi1 and Xili, supporting our observation that Xiwi1, but not Xili, is a nucleo-cytoplasmic protein. Furthermore, by immunoprecipitation of small RNAs, we establish Xiwi1 as a bona fide Piwi protein. These results suggest that the Piwi/piRNA pathway is active in translationally repressed oocytes. This is a significant finding as the Xenopus model provides an excellent tool to study post-transcriptional mechanisms.

Vg1 RBP intracellular distribution and evolutionarily conserved expression at multiple stages during development.

We have analyzed the expression and intracellular distribution, during oogenesis and embryogenesis, of Vg1 RBP, a protein implicated in the intracellular localization of Vg1 mRNA to the vegetal cortex of Xenopus oocytes. Vg1 RBP (protein) colocalizes with Vg1 RNA at all stages of oogenesis. Vg1 RBP RNA, however, localizes to the animal pole during late oogenesis, and remains in the animal blastomeres and ectodermal precursors until its zygotic transcription is activated, around stage 12. Vg1 RBP mRNA then becomes expressed throughout the neural epithelium. Vg1 RBP mRNA expression is also detected in what appears to be neural crest cells undergoing delamination and lateral migration. By tailbud stages, Vg1 RBP expression is present in the branchial arches, otic vesicle, pronephros, and along the neural tube. To examine the expression pattern in different species, we cloned the zebrafish homolog of Vg1 RBP by using a highly homologous EST clone to screen an embryonic cDNA library. In situ hybridization reveals that Vg1 RBP RNA localizes early in oogenesis to the animal pole. Although Vg1 RBP RNA is detected in all blastomeres of the early embryo, the expression pattern in the one day old zebrafish embryo is almost identical to that of the equivalent stage Xenopus embryo. These results indicate that the zygotic expression pattern is similar in frogs and fish, and that there is a conserved zygotic expression of Vg1 RBP distinct from its expression in the oocyte.

The four trypanosomatid eIF4E homologues fall into two separate groups, with distinct features in primary sequence and biological properties

Translation initiation in eukaryotes requires eIF4E, the cap binding protein, which mediates its function through an interaction with the scaffolding protein eIF4G, as part of the eIF4F complex. In trypanosomatids, four eIF4E homologues have been described but the specific function of each is not well characterized. Here, we report a study of these proteins in Trypanosoma brucei (TbEIF4E1 through 4). At the sequence level, they can be assigned to two groups: TbEIF4E1 and 2, similar in size to metazoan eIF4E1; and TbEIF4E3 and 4, with long N-terminal extensions. All are constitutively expressed, but whilst TbEIF4E1 and 2 localize to both the nucleus and cytoplasm, TbEIF4E3 and 4 are strictly cytoplasmic and are also more abundant. After knockdown through RNAi, TbEIF4E3 was the only homologue confirmed to be essential for viability of the insect procyclic form. In contrast, TbEIF4E1, 3 and 4 were all essential for the mammalian bloodstream form. Simultaneous RNAi knockdown of TbEIF4E1 and 2 caused cessation of growth and death in procyclics, but with a delayed impact on translation, whilst knockdown of TbEIF4E3 alone or a combined TbEIF4E1 and 4 knockdown led to substantial translation inhibition which preceded cellular death by several days, at least. Only TbEIF4E3 and 4 were found to interact with T. brucei eIF4G homologues; TbEIF4E3 bound both TbEIF4G3 and 4 whilst TbEIF4E4 bound only to TbEIF4G3. These results are consistent with TbEIF4E3 and 4 having distinct but relevant roles in initiation of protein synthesis.