Building RNA-protein granules: insight from the germline

Abstract

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.

Figure 1

Binding of different protein partners regulates the activity of DEAD-box RNA helicases. (a) Cartoon representation of the DEAD-BOX domains of DBP5 protein (grey): NTD and CTD are N-terminal and C-terminal RecA-like domains, respectively. The bound RNA and ATP are represented using yellow sticks (PDB ID: 3FHT; [14]). (b) Surface representation of the same molecule in the same orientation. The DBP5–NUP214 [14], Me31B–EDC3 [23] and eIF4AIII–MAGOH [21] (PDB IDs: 3FHC, 2WAX, 2HYI respectively)interaction surfaces are mapped on corresponding residues of DBP5 in red, green and cyan, respectively, with potentially overlapping residues mapped in a combination of the relevant colors. The figure highlights the existence of numerous, partially overlapping protein–protein (and protein–RNA)interaction surfaces of the DEAD-box helicase motif and the potential for a large number of alternative regulatory binding events. The inter-molecular surface was defined using the program InsightII (Accelerys), structural alignment of the different DEAD-box helicases was performed using the EBI DALI server [113], and the figures were created using the program Pymol (http://www.pymol.org).

Figure 2

Tudor domains bind to methylated amino acids by using a conserved aromatic cage. A crystal structure of a Tudor domain – sDMA peptide is currently not available. Above is a surface representation of the crystal structure of human germline Tdrd2/Tdrkh Tudor domain with an sDMA-GRG peptide docked in the aromatic cage (adapted from [51]). The protein is in grey, with the side chains of the residues of the cage (L364, Y371, F388, F391)in blue. The peptide is in orange (Gly)and yellow (sDMA)with the Dimethyl-guanidinium group in magenta.

Figure 3

Ping-pong model of transposon silencing by Piwi family proteins in germ granules. Mechanistic details for Drosophila Piwi proteins Aub and Ago3 localized in perinuclear nuage are shown, however, mammals have similar mechanism [71,73,114]. Transposon mRNA exported from the nucleus is recognized by antisense piRNA bound to Aub and then cleaved between nucleotides complementary to nucleotides 10 and 11 of the antisense piRNA. After additional processing of the cleaved RNA, it becomes a sense piRNA which, in association with Ago3, base-pairs with piRNA precursor and, similar to Aub/antisense piRNA complex, cleaves that precursor to generate another antisense piRNA. In mammals, during the ping-pong cycle, antisense and sense piRNAs are bound to Miwi2 and Mili respectively [114]. In addition to transposon mRNA degradation in cytoplasmic germ granules, some Piwi proteins have been implicated in epigenetic silencing of transposon transcription in the nucleus by promoting heterochromatin formation or DNA methylation [73,114].

Figure 4

Building a germ granule. DEAD-box RNA helicases associate with Tud-domain proteins and Piwi proteins. Also, symmetrically dimethylated arginines (sDMAs)of Piwi proteins are recognized by Tud domains. These intermolecular interactions are crucial for the assembly of the functional germ granule structure. While associations between helicases and Piwi proteins and between helicases and Tud-domain proteins have been established, molecular and structural details of these interactions are not known. Helicases reshape RNA–protein complexes (RNA is indicated in green), facilitating post-transcriptional control of expression of germline genes at the level of RNA stability and translation. In addition, helicases may restructure RNAs so that they can provide a scaffold for additional germ granule components, either RNA or proteins. Piwi proteins and piRNAs guard germ cells against transposons which involves recognition of transposon mRNA by antisense piRNA as well as cleavage and eventual degradation of transposon mRNA (Figure 3). Indicated interactions and transposon silencing by Piwi-piRNA complexes have been shown in flies and mammals, demonstrating a remarkable conservation of structural principles of germ granule assembly and function throughout evolution.