Drosophila germ granules are assembled from protein components through different modes of competing interactions with the multi-domain Tudor protein.

Membraneless organelles are RNA-protein assemblies which have been implicated in post-transcriptional control. Germ cells form membraneless organelles referred to as germ granules, which contain conserved proteins including Tudor domain-containing scaffold polypeptides and their partner proteins that interact with Tudor domains. Here, we show that in Drosophila, different germ granule proteins associate with the multi-domain Tudor protein using different numbers of Tudor domains. Furthermore, these proteins compete for interaction with Tudor in vitro and, surprisingly, partition to distinct and poorly overlapping clusters in germ granules in vivo. This partition results in minimization of the competition. Our data suggest that Tudor forms structurally different configurations with different partner proteins which dictate different biophysical properties and phase separation parameters within the same granule.

Looking at the Pretty "Phase" of Membraneless Organelles: A View From Drosophila Glia.

Membraneless granules assemble in different cell types and cellular loci and are the focus of intense research due to their fundamental importance for cellular organization. These dynamic organelles are commonly assembled from RNA and protein components and exhibit soft matter characteristics of molecular condensates currently characterized with biophysical approaches and super-resolution microscopy imaging. In addition, research on the molecular mechanisms of the RNA-protein granules assembly provided insights into the formation of abnormal granules and molecular aggregates, which takes place during many neurodegenerative disorders including Parkinson's diseases (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). While these disorders are associated with formation of abnormal granules, membraneless organelles are normally assembled in neurons and contribute to translational control and affect stability of neuronal RNAs. More recently, a new subtype of membraneless granules was identified in Drosophila glia (glial granules). Interestingly, glial granules were found to contain proteins which are the principal components of the membraneless granules in germ cells (germ granules), indicating some similarity in the functional assembly of these structures in glia and germline. This mini review highlights recent research on glial granules in the context of other membraneless organelles, including their assembly mechanisms and potential functions in the nervous system.

Glial granules contain germline proteins in the Drosophila brain, which regulate brain transcriptome.

Membraneless RNA-protein granules play important roles in many different cell types and organisms. In particular, granules found in germ cells have been used as a paradigm to study large and dynamic granules. These germ granules contain RNA and proteins required for germline development. Here, we unexpectedly identify large granules in specific subtypes of glial cells ("glial granules") of the adult Drosophila brain which contain polypeptides with previously characterized roles in germ cells including scaffold Tudor, Vasa, Polar granule component and Piwi family proteins. Interestingly, our super-resolution microscopy analysis shows that in the glial granules, these proteins form distinct partially overlapping clusters. Furthermore, we show that glial granule scaffold protein Tudor functions in silencing of transposable elements and in small regulatory piRNA biogenesis. Remarkably, our data indicate that the adult brain contains a small population of cells, which express both neuroblast and germ cell proteins. These distinct cells are evolutionarily conserved and expand during aging suggesting the existence of age-dependent signaling. Our work uncovers previously unknown glial granules and indicates the involvement of their components in the regulation of brain transcriptome.

Protein components of ribonucleoprotein granules from Drosophila germ cells oligomerize and show distinct spatial organization during germline development.

The assembly of large RNA-protein granules occurs in germ cells of many animals and these germ granules have provided a paradigm to study structure-functional aspects of similar structures in different cells. Germ granules in Drosophila oocyte's posterior pole (polar granules) are composed of RNA, in the form of homotypic clusters, and proteins required for germline development. In the granules, Piwi protein Aubergine binds to a scaffold protein Tudor, which contains 11 Tudor domains. Using a super-resolution microscopy, we show that surprisingly, Aubergine and Tudor form distinct clusters within the same polar granules in early Drosophila embryos. These clusters partially overlap and, after germ cells form, they transition into spherical granules with the structural organization unexpected from these interacting proteins: Aubergine shell around the Tudor core. Consistent with the formation of distinct clusters, we show that Aubergine forms homo-oligomers and using all purified Tudor domains, we demonstrate that multiple domains, distributed along the entire Tudor structure, interact with Aubergine. Our data suggest that in polar granules, Aubergine and Tudor are assembled into distinct phases, partially mixed at their "interaction hubs", and that association of distinct protein clusters may be an evolutionarily conserved mechanism for the assembly of germ granules.

RNA Selection by PIWI Proteins.

Gene regulation by PIWI-piRNA complexes is determined by the selection of cognate target RNAs by PIWI-piRNA. What are the mechanisms for this selection? There is a rigorous multistep control in identifying target RNAs by PIWI-piRNA structures, and RNA helicases play a potentially crucial role in this process.

An in vivo proteomic analysis of the Me31B interactome in Drosophila germ granules.

Drosophila Me31B is a conserved protein of germ granules, ribonucleoprotein complexes essential for germ cell development. Me31B post-transcriptionally regulates mRNAs by interacting with other germ granule proteins. However, a Me31B interactome is lacking. Here, we use an in vivo proteomics approach to show that the Me31B interactome contains polypeptides from four functional groups: RNA regulatory proteins, glycolytic enzymes, cytoskeleton/motor proteins, and germ plasm components. We further show that Me31B likely colocalizes with the germ plasm components Tudor (Tud), Vasa, and Aubergine in the nuage and germ plasm and provide evidence that Me31B may directly bind to Tud in a symmetrically dimethylated arginine-dependent manner. Our study supports the role of Me31B in RNA regulation and suggests its novel roles in germ granule assembly and function.

In vivo mapping of a dynamic ribonucleoprotein granule interactome in early Drosophila embryos.

Macromolecular complexes and organelles play crucial roles within cells, but their native architectures are often unknown. Here, we use an evolutionarily conserved germline organelle, the germ granule, as a paradigm. In Drosophila embryos, we map one of its interactomes using a novel in vivo crosslinking approach that employs two interacting granule proteins and determines their common neighbor molecules. We identified an in vivo granule assembly of Tudor, Aubergine, motor and metabolic proteins, and RNA helicases, and provide evidence for direct interactions within this assembly using purified components. Our study indicates that germ granules contain efficient biochemical reactors involved in post-transcriptional gene regulation.

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.

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.

Next generation organelles: structure and role of germ granules in the germline.

Germ cells belong to a unique class of stem cells that gives rise to eggs and sperm, and ultimately to an entire organism after gamete fusion. In many organisms, germ cells contain electron-dense structures that are also known as nuage or germ granules. Although germ granules were discovered more than 100 years ago, their composition, structure, assembly, and function are not fully understood. Germ granules contain non-coding RNAs, mRNAs, and proteins required for germline development. Here we review recent studies that highlight the importance of several protein families in germ granule assembly and function, including germ granule inducers, which initiate the granule formation, and downstream components, such as RNA helicases and Tudor domain-Piwi protein-piRNA complexes. Assembly of these components into one granule is likely to result in a highly efficient molecular machine that ensures translational control and protects germline DNA from mutations caused by mobile genetic elements. Furthermore, recent studies have shown that different somatic cells, including stem cells and neurons, produce germ granule components that play a crucial role in stem cell maintenance and memory formation, indicating a much more diverse functional repertoire for these organelles than previously thought.

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.

Building RNA-protein granules: insight from the germline.

The germline originates from primordial embryonic germ cells which give rise to sperm and egg cells and consequently, to the next generation. Germ cells of many organisms contain electron-dense granules that comprise RNA and proteins indispensable for germline development. Here we review recent reports that provide important insights into the structure and function of crucial RNA and protein components of the granules, including DEAD-box helicases, Tudor domain proteins, Piwi/Argonaute proteins and piRNA. Collectively, these components function in translational control, remodeling of ribonucleoprotein complexes and transposon silencing. Furthermore, they interact with each other by means of conserved structural modules and post-translationally modified amino acids. These data suggest a widespread use of several protein motifs in germline development and further our understanding of other ribonucleoprotein structures, for example, processing bodies and neuronal granules.

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.

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.

Mutational eidence for a functional connection between two domains of 23S rRNA in translation termination.

Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.