Germ Plasm Biogenesis--An Oskar-Centric Perspective

Abstract

Germ granules are the hallmark of all germ cells. These membrane-less, electron-dense structures were first observed over 100 years ago. Today, their role in regulating and processing transcripts critical for the establishment, maintenance, and protection of germ cells is well established, and pathways outlining the biochemical mechanisms and physical properties associated with their biogenesis are emerging.

Keywords: Drosophila; Germ cells; Germ granules; Germ plasm; Nanos; Nuage; Oskar; Phase transition; RNA granules; RNA localization; RNA translation; Tudor; Vasa.

Figure 1

Establishment of anterior-posterior polarity during Drosophila oogenesis a. Ovariole with developing egg chambers. Each ovary contains 12–16 ovarioles. At the tip (left, b-c region) is the germarium, where 2–3 GSCs generate a cytoblast that matures into a egg chamber consisting of the oocyte and 15 nurse cells surrounded by somatic follicle cells (stage 1 egg chamber). Daily stem cell division produces a chain of maturing egg chambers that mature to the fully developed egg (right, g). Small letters on top refer to stages depicted in other figure panels. b. Division pattern of the cystoblast, the GSC daughter in the germarium. After division cells remain connected via ring canals with fusome material traversing the cyst. The first two cells born are connected by four ring canals, the next two by three, the next four by two and the last 8 by one ring canal each. Note that the earlier born cells inherit proportionally more fusome material. c. 16 cell cyst in germarium, all cells are connected by ring canals, and fusomes. The two cells with four ring canals “pro-oocytes” are positioned posteriorly within the cluster and enter into meiosis (left), only one of the two cells remains in meiosis and arrests in meiotic prophase (right). All other cells develop into polyploid nurse cells, which continue to be connected to the oocyte by ring canals. All centrosomes migrate into the oocyte and a microtubule network nucleate from the oocyte to the nurse cells. Dynein mediated transport brings cargo and RNAs from the nurse cell into the oocyte. Densely packed mitochondria and RNA form the Balbiani body (yellow in c and c’). c’ oocyte cytoplasm indicating microtubule polarity and RNA transport particles (after (Roth and Lynch, 2009)). d. Stage 5 egg chamber. Nurse cells and oocyte are surrounded by somatic follicle cells. Continued dynein-mediated RNA transport from the nurse cells into oocyte. Microtubules are oriented to nucleate (- pole) from oocyte and extend into nurse cells. Nuage: RNA rich granules form around nurse cell nucleus often in close proximity of nuclear pore. e. Stage 10A egg chamber. Mutually exclusive interactions between Par-1 (Blue) at the posterior pole and the Par-3/Bazooka (yellow) complex at the lateral and anterior cortex establish polarity within the oocyte. Microtubules nucleate from lateral and anterior cortex extending into the oocyte and are inhibited at the posterior pole by Par-1. Kinesin motors transport osk RNA to the posterior pole, where it is translated. Dynein-mediated transport continues to bring osk RNA from the nurse cells to the oocyte. Somatic follicle cells surround the egg chamber. e’ Summary of microtubule organization and osk RNA transport dynamics during stage 10A. Microtubule network is denser at the anterior than posterior of oocyte (green). A slight bias of kinesin-mediated microtubule transport (red line) leads to enrichment of osk RNA at posterior pole over time (red stippled) (after (Parton et al., 2011)). f. Stage 10B/11, cytoplasmic streaming. The actin meshwork is disassembled in the oocyte and microtubules align in bundles (green). This leads to a streaming process where RNAs and proteins are mixed within the oocyte and additional RNAs/proteins are drawn into the oocyte (green arrow). Effector RNAs like nanos (red) become tethered at posterior pole. g. Mature egg. Germplasm is assembled and polar granules of similar size to mitochondria are found in a precise region at the posterior pole. Effector RNAs are localized to the granules, Osk, Vasa, Tud and Aub proteins are essential part of the granules but osk RNA is not associated with polar granules (see Fig 3E).

Figure 2

Oskar RNA and protein history a. Structure of nascent osk RNA. Exon junction complex components bind to sequences flanking the first intron, which is critical for kinesin-mediated osk RNA transport. b. Osk RNA transport particles are transported along microtubule by dynein (directed towards – end of microtubule) and kinesin (directed towards + end of microtubule) motors. The oocyte entry signal (d) regulates the dynein-mediated transport of osk RNA into the early oocyte. (c) The Spliced Oskar Localization Element (SOLE) forms as a consequence of first intron splicing, where 18nt from the first and 10 nt from the second exon merge into a stem loop strcuture. The DD palindromic sequence element (d) is located in the loop of the OES stem loop. Via RNA-RNA “kissing interaction” this element promotes RNA transport particle formation and hitchhiking of RNA lacking the SOLE element on those with the element. AB, C region are regions rich of Bruno regulatory elements (BRE), which repress RNA translation, and IMP binding elements, which promote translation and localization. Binding elements close to the most distal part of the 3′UTR are important for translational activation, via Adenine-rich sequences (ARS) that mediate polyadenylation. e. Structure of Oskar isoforms. Left: Long Oskar is a 606 aminoacid protein, the N-terminal extension (NTE) is required for actin filament nucleation and enhanced endocytosis at the posterior pole. Somehow with NTE suppresses short-Oskar functions. Right: Short -Oskar start at the second Methionine 139. The Lotus domain dimerizes and interacts with Vasa (see also g), the C-terminal OSK domain resembles a SGNH hydrolase domain, however the enzymatic amino acids are missing. Instead the OSK domain is thought to interact with effector RNAs. f. Structure diagram of Lotus and OSK domain. The Lotus domain forms a homodimer of two winged helix structures and interacts with Vasa, while the OSK domain forms a globular structure with basic and hydrophilic aminoacids exposed to the surface, which are thought to interact with RNA.

Figure 3

Germ plasm assembly a. Multiple controls described in Fig 1 and 2 feed into the localization and translation of osk. Osk protein recruits other components of the germplasm such as Vasa, Tudor and Aubergine. Symmetric dimethylation of Arginine (sDMA) in Vasa and Aubergine by the methyltransferase Capsuleen allow binding to Tudor domain proteins (Kirino et al., 2010a; Kirino et al., 2010b; Liu et al., 2010; Webster et al., 2015). This together with liquid to gel phase transitions could provide a possible basis for the dense assembly of polar granules. b. Model for phase transition that occurs at posterior pole when concentration of germ plasm protein and RNAs become high (Brangwynne, 2013). c. SIM image of nos and gcl RNA hybridization using Stellaris© RNA in situ hybridization probes. C’ higher magnification to reveal particle overlap. d. Germ plasm with polar granules marked by Vasa (filled arrow) and mitochondria (open arrow). e. Organization of homotypic RNA clusters within polar granules according to (Trcek et al., 2015).