Structure and function of eTudor domain containing TDRD proteins

Tudor domain-containing (TDRD) proteins, as a family of evolutionarily conserved proteins, have been studied extensively in recent years in terms of their biological and biochemical functions. A major function of the TDRD proteins is to recognize the N-terminal arginine-rich motifs of the P-element-induced wimpy testis (PIWI) proteins via their conserved extended Tudor (eTudor or eTud) domains, which is essential in piRNA biogenesis and germ cell development. In this review, we summarize recent progress in the study of the TDRD proteins, and discuss the molecular mechanisms for the different binding selectivity of these eTudor domains to PIWI proteins based on the available binding and structural data. Understanding the binding differences of these TDRDs to PIWI proteins will help us better understand their functional differences and aid us in developing the target-specific therapeutics, because overexpression or mutations of the human TDRD proteins have been demonstrated to associate with various diseases.     

Mouse Tudor Repeat-1 (MTR-1) is a novel component of chromatoid bodies/nuages in male germ cells and forms a complex with snRNPs

Characteristic ribonucleoprotein-rich granules, called nuages, are present in the cytoplasm of germ-line cells in many species. In mice, nuages are prominent in postnatal meiotic spermatocytes and postmeiotic round spermatids, and are often called chromatoid bodies at the stages. We have isolated Mouse tudor repeat-1 (Mtr-1) which encodes a MYND domain and four copies of the tudor domain. Multiple tudor domains are a characteristic of the TUDOR protein, a component of Drosophila nuages. Mtr-1 is expressed in germ-line cells and is most abundant in fetal prospermatogonia and postnatal primary spermatocytes. The MTR-1 protein is present in the cytoplasm of prospermatogonia, spermatocytes, and round spermatids, and predominantly localizes to chromatoid bodies. We show that (1) an assembled form of small nuclear ribonucleoproteins (snRNPs), which usually function as spliceosomal complexes in the nucleus, accumulate in chromatoid bodies, and form a complex with MTR-1, (2) when expressed in cultured cells, MTR-1 forms discernible granules that co-localize with snRNPs in the cell plasm during cell division, and (3) the deletion of multiple tudor domains in MTR-1 abolishes the formation of such granules. These results suggest that MTR-1, which would provide novel insights into evolutionary comparison of nuages, functions in assembling snRNPs into cytoplasmic granules in germ cells.     

1H, 15N and 13C resonance assignments for the three LOTUS RNA binding domains of Tudor domain-containing protein TDRD7

The LOTUS or OST-HTH domain is a recently discovered motif of about 80 amino acids and is found in several germline-specific proteins including the Tudor domain-containing proteins TDRD5 and TDRD7, which are important for germ cell development. The LOTUS domain is an RNA binding domain but its exact function is unknown. Here, we report the ¹H, ¹³C and ¹⁵N resonance assignments for the three LOTUS domains present in mouse TDRD7. These assignments will allow three-dimensional structure determination of the LOTUS domains and mapping of their interaction with RNA, steps toward deciphering the function of TDRD7.     

Sm proteins, the constituents of the spliceosome, are components of nuage and mitochondrial cement in Xenopus oocytes

A conserved feature of germ cells in many animal species is the presence of perinuclear electron-dense material called the "nuage" that is believed to be a precursor of germinal (or polar or P) granules. In Xenopus oogenesis the nuage is first observed near the nuclear envelope and subsequently in close contact with mitochondria, at which stage it is called the mitochondrial cement. In this study, we found that, in Xenopus pre-stage I and stage I oocytes, nuage and mitochondrial cement contain the spliceosomal Sm proteins, Xcat2 mRNA, and DEAD-box RNA helicase XVLG1. Other components of Cajal bodies or splicing machinery such as coilin, SMN protein, and snRNAs are absent from the nuage and mitochondrial cement. We suggest that Xenopus Sm proteins have adapted to a role independent of pre-mRNA splicing and that instead of binding to their traditional spliceosomal partner such as snRNA, they bind mRNAs that are the components of germinal granules (i.e., Xcat2 mRNA) and facilitate the transport of these mRNAs from the nucleus to the nuage that is a precursor of germinal granules. In addition, the presence of Vasa-like DEAD-box helicase in Xenopus nuage suggests involvement of nuage in the microRNA and/or RNAi pathway, similar to the role of nuage in Drosophila.     

The role of Tudor domains in germline development and polar granule architecture

Tudor domains are found in many organisms and have been implicated in protein-protein interactions in which methylated protein substrates bind to these domains. Here, we present evidence for the involvement of specific Tudor domains in germline development. Drosophila Tudor, the founder of the Tudor domain family, contains 11 Tudor domains and is a component of polar granules and nuage, electron-dense organelles characteristic of the germline in many organisms, including mammals. In this study, we investigated whether the 11 Tudor domains fulfil specific functions for polar granule assembly, germ cell formation and abdomen formation. We find that even a small number of non-overlapping Tudor domains or a substantial reduction in overall Tudor protein is sufficient for abdomen development. In stark contrast, we find a requirement for specific Tudor domains in germ cell formation, Tudor localization and polar granule architecture. Combining genetic analysis with structural modeling of specific Tudor domains, we propose that these domains serve as ;docking platforms' for polar granule assembly.     

TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution

R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic ‘reader’ Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.     

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.     

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.     

Spindle-E cycling between nuage and cytoplasm is controlled by Qin and PIWI proteins

Transposable elements (TEs) are silenced in germ cells by a mechanism in which PIWI proteins generate and use PIWI-interacting ribonucleic acid (piRNA) to repress expression of TE genes. piRNA biogenesis occurs by an amplification cycle in microscopic organelles called nuage granules, which are localized to the outer face of the nuclear envelope. One cofactor required for amplification is the helicase Spindle-E (Spn-E). We found that the Spn-E protein physically associates with the Tudor domain protein Qin and the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3). Spn-E and Qin proteins are mutually dependent for their exit from nuage granules, whereas Spn-E and both Aub and Ago3 are mutually dependent for their entry or retention in nuage. The result is a dynamic cycling of Spn-E and its associated factors in and out of nuage granules. This implies that nuage granules can be considered to be hubs for active, mobile, and transient complexes. We suggest that this is in some way coupled with the execution of the piRNA amplification cycle.     

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.     

nanos1 is required to maintain oocyte production in adult zebrafish

Development of the germline requires the specification and survival of primordial germ cells (PGCs) in the embryo as well as the maintenance of gamete production during the reproductive life of the adult. These processes appear to be fundamental to all Metazoans, and some components of the genetic pathway regulating germ cell development and function are evolutionarily conserved. In both vertebrates and invertebrates, nanos-related genes, which encode RNA-binding zinc finger proteins, have been shown to play essential and conserved roles during germ cell formation. In Drosophila, maternally supplied nanos is required for survival of PGCs in the embryo, while in adults, nanos is required for the continued production of oocytes by maintaining germline stem cells self-renewal. In mice and zebrafish, nanos orthologs are required for PGC survival during embryogenesis, but a role in adults has not been explored. We show here that nanos1 in zebrafish is expressed in early stage oocytes in the adult female germline. We have identified a mutation in nanos1 using a reverse genetics method and show that young female nanos mutants contain oocytes, but fail to maintain oocyte production. This progressive loss of fertility in homozygous females is not a phenotype that has been described previously in the zebrafish and underlines the value of a reverse genetics approach in this model system.     

OST-HTH: a novel predicted RNA-binding domain

Background  

The mechanism by which the arthropod Oskar and vertebrate TDRD5/TDRD7 proteins nucleate or organize structurally related ribonucleoprotein (RNP) complexes, the polar granule and nuage, is poorly understood. Using sequence profile searches we identify a novel domain in these proteins that is widely conserved across eukaryotes and bacteria.