Novel role of specific Tudor domains in Tudor-Aubergine protein complex assembly and distribution during Drosophila oogenesis

Germ cells give rise to the next generation and contain ribonucleoprotein particles, germ granules. In these granules, Piwi protein Aubergine has been shown to interact with Tudor protein in Drosophila. Tudor protein has 11 Tudor domains and it has been unclear to what extent all these domains are involved in the interaction with Aubergine. Here we present direct biochemical evidence that Tudor-Aubergine interaction surface is composed of different Tudor domains including those that have not been previously implicated in Aubergine recognition. Furthermore, we show that specific single Tudor domains determine localization of Tudor complex to different sites in ovarian germ cells. Our data suggest that multiple Tudor domains of germline proteins from various species are redundantly used for interaction with the same protein partner during germline development.     

Tudor and its domains： germ cell formation from a Tudor perspective

In many metazoan species, germ cell formation requires the germ plasm, a specialized cytoplasm which often contains electron dense structures. Genes required for germ cell formation in Drosophila have been isolated predominantly in screens for maternal-effect mutations. One such gene is tudor (tud); without proper tud function germ cell formation does not occur. Unlike other genes involved in Drosophila germ cell specification tud is dispensable for other somatic functions such as abdominal patterning. It is not known how TUD contributes at a molecular level to germ cell formation but in tud mutants, polar granule formation is severely compromised, and mitochondrially encoded ribosomal RNAs do not localize to the polar granule. TUD is composed of 11 repeats of the protein motif called the Tudor domain. There are similar proteins to TUD in the germ line of other metazoan species including mice. Probable vertebrate orthologues of Drosophila genes involved in germ cell specification will be discussed.     

Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins

Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components we isolated proteins that associate in early embryos with Vasa (VAS) and Tudor (TUD), two known polar granule associated molecules. We identified Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) as components of both VAS and TUD complexes and confirmed their localization to polar granules by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. Our recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly.     

Targeting and anchoring Tudor in the pole plasm of the Drosophila oocyte

Background

Germline formation is a highly regulated process in all organisms. In Drosophila embryos germ cells are specified by the pole plasm, a specialized cytoplasmic region containing polar granules. Components of these granules are also present in the perinuclear ring surrounding nurse cells, the nuage. Two such molecules are the Vasa and Tudor proteins. How Tudor localizes and is maintained in the pole plasm is, however, not known.

Methodology/Principal Findings

Here, the process of Tudor localization in nuage and pole plasm was analyzed. The initial positioning of Tudor at the posterior pole of stage 9 oocytes was found to occur in the absence of a structurally detectable nuage. However, in mutants for genes encoding components of the nuage, including vasa, aubergine, maelstrom, and krimper, Tudor was detached from the posterior cortex in stage 10 oocytes, suggesting a prior passage in the nuage for its stability in the pole plasm. Further studies indicated that Valois, which was previously shown to bind in vitro to Tudor, mediates the localization of Tudor in the pole plasm by physically interacting with Oskar, the polar granule organizer. An association between Tudor and Vasa mediated by RNA was also detected in ovarian extracts.

Conclusions/Significance

The present data challenge the view that the assembly of the polar granules occurs in a stepwise and hierarchical manner and, consequently, a revised model of polar granule assembly is proposed. In this model Oskar recruits two downstream components of the polar granules, Vasa and Tudor, independently from each other: Vasa directly interacts with Oskar while Valois mediates the recruitment of Tudor by interacting with Oskar and Tudor.     

The role of mitochondrial rRNAs and nanos protein in germline formation in Drosophila embryos

Germ cells, represented by male sperm and female eggs, are specialized cells that transmit genetic material from one generation to the next during sexual reproduction. The mechanism by which multicellular organisms achieve the proper separation of germ cells and somatic cells is one of the longest standing issues in developmental biology. In many animal groups, a specialized portion of the egg cytoplasm, or germ plasm, is inherited by the cell lineage that gives rise to the germ cells (germline). Germ plasm contains maternal factors that are sufficient for germline formation. In the fruit fly, Drosophila, germ plasm is referred to as polar plasm and is distinguished histologically by the presence of polar granules, which act as a repository for the maternal factors required for germline formation. Molecular screens have so far identified several of these factors that are enriched in the polar plasm. This article focuses on the molecular functions of two such factors in Drosophila, mitochondrial ribosomal RNAs and Nanos protein, which are required for the formation and differentiation of the germline progenitors, respectively.     

Mitochondrial trafficking through Rhot1 is involved in the aggregation of germinal granule components during primordial germ cell formation in Xenopus embryos

In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline‐Mt). However, the role of germline‐Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline‐Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C‐terminal mitochondrial transmembrane domain, inhibited the aggregation of germline‐Mt during early development. In Rhot1‐inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail‐bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.     

Arginine methyltransferase Capsuleen is essential for methylation of spliceosomal Sm proteins and germ cell formation in Drosophila

Although arginine modification has been implicated in a number of cellular processes, the in vivo requirement of protein arginine methyltransferases (PRMTs) in specific biological processes remain to be clarified. In this study we characterize the Drosophila PRMT Capsuléen, homologous to human PRMT5. During Drosophila oogenesis, catalytic activity of Capsuléen is necessary for both the assembly of the nuage surrounding nurse cell nuclei and the formation of the pole plasm at the posterior end of the oocyte. In particular, we show that the nuage and pole plasm localization of Tudor, an essential component for germ cell formation, are abolished in csul mutant germ cells. We identify the spliceosomal Sm proteins as in vivo substrates of Capsuléen and demonstrate that Capsuléen, together with its associated protein Valois, is essential for the synthesis of symmetric di-methylated arginyl residues in Sm proteins. Finally, we show that Tudor can be targeted to the nuage in the absence of Sm methylation by Capsuléen, indicating that Tudor localization and Sm methylation are separate processes. Our results thus reveal the role of a PRMT in protein localization in germ cells.     

C-terminal moiety of Tudor contains its in vivo activity in Drosophila

Background

In early Drosophila embryos, the germ plasm is localized to the posterior pole region and is partitioned into the germline progenitors, known as pole cells. Germ plasm, or pole plasm, contains the polar granules which form during oogenesis and are required for germline development. Components of these granules are also present in the perinuclear region of the nurse cells, the nuage. One such component is Tudor (Tud) which is a large protein containing multiple Tudor domains. It was previously reported that specific Tudor domains are required for germ cell formation and Tud localization.

Methodology/Principal Findings

In order to better understand the function of Tud the distribution and functional activity of fragments of Tud were analyzed. These fragments were fused to GFP and the fusion proteins were synthesized during oogenesis. Non-overlapping fragments of Tud were found to be able to localize to both the nuage and pole plasm. By introducing these fragments into a tud mutant background and testing their ability to rescue the tud phenotype, I determined that the C-terminal moiety contains the functional activity of Tud. Dividing this fragment into two parts reduces its localization in pole plasm and abolishes its activity.

Conclusions/Significance

I conclude that the C-terminal moiety of Tud contains all the information necessary for its localization in the nuage and pole plasm and its pole cell-forming activity. The present results challenge published data and may help refining the functional features of Tud.     

Developmentally regulated expression of mil-1 and mil-2, mouse interferon-induced transmembrane protein like genes,during formation and differentiation of primordial germ cells

In all multicellular organisms, germ cells originating from a fertilized egg have the highly specialized role of transmitting genetic information to the next generation. In many animal species, the establishment of the germ cell lineage is regulated by the maternally inherited germplasm. In mammals, however, germline determination is not based on the unequal distribution of maternal determinants. In the processes of mammalian germ cell formation and subsequent differentiation, the molecular basis of the acquisition of germ cell status is not well understood. Since migrating primordial germ cells (PGCs) are lineage-restricted to the germline, they have already acquired a germ cell specific fate distinct from that of pluri/multi-potent stem cells. However, there have been no molecules known to be expressed in migrating PGCs but not in the inner cell mass of blastocysts. Such molecules should be involved in early germ cell development, and they should make good markers for following the process of PGC formation. To identify such molecules, we performed a subtracted cDNA screening with migrating PGCs and blastocysts in mice, and isolated 11 clones preferentially expressed in PGCs. Here, we report the identification of two genes with similarity to human interferon-induced transmembrane protein (Ifitm) genes, and expression patterns of these genes in forming and in differentiating PGCs. During germ cell formation, mouse Ifitm like (mil)-1 was expressed in putative PGC ancestors in embryos at 6.5-7.5 days post coitum. In migrating PGCs, mil-1 expression was continuously observed and mil-2 expression was first detected during germ cell differentiation.     

Developmentally regulated expression of mil-1 and mil-2, mouse interferon-induced transmembrane protein like genes, during formation and differentiation of primordial germ cells

In all multicellular organisms, germ cells originating from a fertilized egg have the highly specialized role of transmitting genetic information to the next generation. In many animal species, the establishment of the germ cell lineage is regulated by the maternally inherited germplasm. In mammals, however, germline determination is not based on the unequal distribution of maternal determinants. In the processes of mammalian germ cell formation and subsequent differentiation, the molecular basis of the acquisition of germ cell status is not well understood. Since migrating primordial germ cells (PGCs) are lineage-restricted to the germline, they have already acquired a germ cell specific fate distinct from that of pluri/multi-potent stem cells. However, there have been no molecules known to be expressed in migrating PGCs but not in the inner cell mass of blastocysts. Such molecules should be involved in early germ cell development, and they should make good markers for following the process of PGC formation. To identify such molecules, we performed a subtracted cDNA screening with migrating PGCs and blastocysts in mice, and isolated 11 clones preferentially expressed in PGCs. Here, we report the identification of two genes with similarity to human interferon-induced transmembrane protein (Ifitm) genes, and expression patterns of these genes in forming and in differentiating PGCs. During germ cell formation, mouse Ifitm like (mil)-1 was expressed in putative PGC ancestors in embryos at 6.5-7.5 days post coitum. In migrating PGCs, mil-1 expression was continuously observed and mil-2 expression was first detected during germ cell differentiation.     

Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ cells,bind Tudor and protect from transposable elements

Germ cells give rise to all cell lineages in the next‐generation and are responsible for the continuity of life. In a variety of organisms, germ cells and stem cells contain large ribonucleoprotein granules. Although these particles were discovered more than 100 years ago, their assembly and functions are not well understood. Here we report that glycolytic enzymes are components of these granules in Drosophila germ cells and both their mRNAs and the enzymes themselves are enriched in germ cells. We show that these enzymes are specifically required for germ cell development and that they protect their genomes from transposable elements, providing the first link between metabolism and transposon silencing. We further demonstrate that in the granules, glycolytic enzymes associate with the evolutionarily conserved Tudor protein. Our biochemical and single‐particle EM structural analyses of purified Tudor show a flexible molecule and suggest a mechanism for the recruitment of glycolytic enzymes to the granules. Our data indicate that germ cells, similarly to stem cells and tumor cells, might prefer to produce energy through the glycolytic pathway, thus linking a particular metabolism to pluripotency.     

The oyster vasa-like gene: a specific marker of the germline in Crassostrea gigas

The vasa gene is a key determinant for germline formation in eukaryotes. This gene, highly conserved through evolution, encodes a RNA helicase protein member of the DEAD-box family. To understand the germline formation in oyster, we report here the isolation and the characterization of a vasa orthologue in Crassostrea gigas (Oyvlg). OyVLG contained the eight consensus domains of the DEAD-box including those providing RNA unwinding activity. The expression pattern of Oyvlg was examined in adult oyster tissues at different reproductive stages. Its expression was restricted to germline cells both in males and females, including germinal stem cells and auxiliary cells. The expression of Oyvlg, strongest in early germ cells, decreased as the maturation proceeded. These data and the evolutionary conservation observed suggested the role of Oyvlg in germline development. Oyvlg is the first germ cell specific marker in oyster and will be very useful in studies of oyster germline formation.     

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.     

Tudor-related proteins TDRD1/MTR-1, TDRD6 and TDRD7/TRAP: domain composition, intracellular localization, and function in male germ cells in mice

The germ-line cells of many animals possess a characteristic cytoplasmic structure termed nuage or germinal granules. In mice, nuage that is prominent in postnatal male germ cells is also called intermitochondrial cement or chromatoid bodies. TDRD1/MTR-1, which contains Tudor domain repeats, is a specific component of the mouse nuage, analogously to Drosophila Tudor, a constituent of polar granules/nuage in oocytes and embryos. We show that TDRD6 and TDRD7/TRAP, which also contain multiple Tudor domains, specifically localize to nuage and form a ribonucleoprotein complex together with TDRD1/MTR-1. The characteristic co-localization of TDRD1, 6 and 7 was disrupted in a mutant of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), which encodes another evolutionarily conserved component of nuage. In vivo over-expression experiments of the TDRD proteins and truncated forms during male germ cell differentiation showed that a single Tudor domain is a structural unit that localizes or accumulates to nuage, but the expression of the truncated, putative dominant negative forms is detrimental to meiotic spermatocytes. These results indicate that the Tudor-related proteins, which contain multiple repeats of the Tudor domain, constitute an evolutionarily conserved class of nuage components in the germ-line, and their localization or accumulation to nuage is likely conferred by a Tudor domain structure and downstream of Mvh, while the characteristic repeated architecture of the domain is functionally essential for the differentiation of germ cells.     

Deciphering arginine methylation: Tudor tells the tale

Proteins can be modified by post-translational modifications such as phosphorylation, methylation, acetylation and ubiquitylation, creating binding sites for specific protein domains. Methylation has pivotal roles in the formation of complexes that are involved in cellular regulation, including in the generation of small RNAs. Arginine methylation was discovered half a century ago, but the ability of methylarginine sites to serve as binding motifs for members of the Tudor protein family, and the functional significance of the protein-protein interactions that are mediated by Tudor domains, has only recently been appreciated. Tudor proteins are now known to be present in PIWI complexes, where they are thought to interact with methylated PIWI proteins and regulate the PIWI-interacting RNA (piRNA) pathway in the germ line.     

PKA-R1 spatially restricts Oskar expression for Drosophila embryonic patterning

Targeting proteins to specific domains within the cell is central to the generation of polarity, which underlies many processes including cell fate specification and pattern formation during development. The anteroposterior and dorsoventral axes of the Drosophila melanogaster embryo are determined by the activities of localized maternal gene products. At the posterior pole of the oocyte, Oskar directs the assembly of the pole plasm, and is thus responsible for formation of abdomen and germline in the embryo. Tight restriction of oskar activity is achieved by mRNA localization, localization-dependent translation, anchoring of the RNA and protein, and stabilization of Oskar at the posterior pole. Here we report that the type 1 regulatory subunit of cAMP-dependent protein kinase (Pka-R1) is crucial for the restriction of Oskar protein to the oocyte posterior. Mutations in PKA-R1 cause premature and ectopic accumulation of Oskar protein throughout the oocyte. This phenotype is due to misregulation of PKA catalytic subunit activity and is suppressed by reducing catalytic subunit gene dosage. These data demonstrate that PKA mediates the spatial restriction of Oskar for anteroposterior patterning of the Drosophila embryo and that control of PKA activity by PKA-R1 is crucial in this process.     

Germ plasm in Caenorhabditis elegans, Drosophila and Xenopus

Special cytoplasm, called germ plasm, that is essential for the differentiation of germ cells is localized in a particular region of Caenorhabditis elegans, Drosophila and Xenopus eggs. The mode of founder cell formation of germline, the origin and behavior of the germline granules, and the molecules localized in germline cells are compared in these organisms. The common characteristics of the organisms are mainly as follows. First, the founder cells of germline are established before the intiation of gastrulation. Second, the germline granules or their derivatives are always present in germline cells or germ cells throughout the life cycle in embryos, larvae, and adults. Lastly, among the proteins localized in the germ plasm, only Vasa protein or its homolog is detected in the germline cells or germ cells throughout the life cycle. As the protein of vasa homolog has been reported to be also localized in the germline-specific structure or nuage in some of the organisms without the germ plasm, the possibility that the mechanism for differentiation of primordial germ cells is basically common in all organisms with or without the germ plasm is discussed.     

A Highlights from MBoC Selection: DLC-1 facilitates germ granule assembly in Caenorhabditis elegans embryo

Germ granules are cytoplasmic assemblies of RNA-binding proteins (RBPs) required for germ cell development and fertility. During the first four cell divisions of the Caenorhabditis elegans zygote, regulated assembly of germ (P) granules leads to their selective segregation to the future germ cell. Here we investigate the role of DLC-1, a hub protein implicated in stabilization and function of diverse protein complexes, in maintaining P granule integrity. We find that DLC-1 directly interacts with several core P granule proteins, predominantly during embryogenesis. The loss of dlc-1 disrupts assembly of P granule components into phase-separated organelles in the embryos, regardless of whether or not DLC-1 directly interacts with these proteins. Finally, we infer that P granule dispersal in the absence of dlc-1 is likely independent of DLC-1’s function as a subunit of the dynein motor and does not result from a loss of cell polarity.     

Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization

Piwi proteins such as Drosophila Aubergine (Aub) and mouse Miwi are essential for germline development and for primordial germ cell (PGC) specification. They bind piRNAs and contain symmetrically dimethylated arginines (sDMAs), catalyzed by dPRMT5. PGC specification in Drosophila requires maternal inheritance of cytoplasmic factors, including Aub, dPRMT5, and Tudor (Tud), that are concentrated in the germ plasm at the posterior end of the oocyte. Here we show that Miwi binds to Tdrd6 and Aub binds to Tudor, in an sDMA-dependent manner, demonstrating that binding of sDMA-modified Piwi proteins with Tudor-domain proteins is an evolutionarily conserved interaction in germ cells. We report that in Drosophila tud¹ mutants, the piRNA pathway is intact and most transposons are not de-repressed. However, the localization of Aub in the germ plasm is severely reduced. These findings indicate that germ plasm assembly requires sDMA modification of Aub by dPRMT5, which, in turn, is required for binding to Tudor. Our study also suggests that the function of the piRNA pathway in PGC specification may be independent of its role in transposon control.