Extended Tudor Domain Research Articles

Tdrd1 is a germline-specific and sexually dimorphically expressed gene in Paralichthys olivaceus

Tudor domain containing protein 1 (tdrd1) is a member of the Tudor family and has shown essential functions during embryogenesis and gametogenesis. In this study, we cloned the full length cDNA of Paralichthys olivaceus tdrd1 (Potdrd1). PoTDRD1 is a multidomain protein with an N-terminal MYND zinc finger domain, followed by four tandem extended Tudor domains. Sequence comparison, genomic structure, phylogenetic analyses and synteny analyses showed that Potdrd1 was homologous to those of other teleosts. In adult individuals, the expression of Potdrd1 was higher in testis than in ovary, demonstrating a sexually dimorphic gene expression pattern. In situ hybridization (ISH) showed that Potdrd1 mRNA was detected in oogonia and oocytes of ovary as well as in spermatogonia and spermatocytes of testis. In juveniles during gonad differentiation its expression level increased rapidly from 30 dph to 100 dph and showed obvious sexual dimorphism that was in accordance with the expression of anti-Mullerian hormone (amh). Potdrd1 mRNA was consistently detected during embryogenesis, and its level was higher from unfertilzed eggs to the blastula stage and subsequently decreased until hatching. When chimeric RNA containing green fluorescent protein (GFP) and 3′ untranslated regions (UTR) of Potdrd1 was microinjected into zebrafish fertilized eggs, the green fluorescence could be visualized only in putative PGCs. These results indicated that Potdrd1 is a germline specific and sexually dimorphic factor that potentially functionate in germline development and gametogenesis in Japanese flounder.

Structural basis for arginine methylation-independent recognition of PIWIL1 by TDRD2

The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway plays a central role in transposon silencing and genome protection in the animal germline. A family of Tudor domain proteins regulates the piRNA pathway through direct Tudor domain-PIWI interactions. Tudor domains are known to fulfill this function by binding to methylated PIWI proteins in an arginine methylation-dependent manner. Here, we report a mechanism of methylation-independent Tudor domain-PIWI interaction. Unlike most other Tudor domains, the extended Tudor domain of mammalian Tudor domain-containing protein 2 (TDRD2) preferentially recognizes an unmethylated arginine-rich sequence from PIWI-like protein 1 (PIWIL1). Structural studies reveal an unexpected Tudor domain-binding mode for the PIWIL1 sequence in which the interface of Tudor and staphylococcal nuclease domains is primarily responsible for PIWIL1 peptide recognition. Mutations disrupting the TDRD2-PIWIL1 interaction compromise piRNA maturation via 3'-end trimming in vitro. Our work presented here reveals the molecular divergence of the interactions between different Tudor domain proteins and PIWI proteins.

The multiple Tudor domain-containing protein TDRD1 is a molecular scaffold for mouse Piwi proteins and piRNA biogenesis factors

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs expressed in the germline of animals. They associate with Argonaute proteins of the Piwi subfamily, forming ribonucleoprotein complexes that are involved in maintaining genome integrity. The N-terminal region of some Piwi proteins contains symmetrically dimethylated arginines. This modification is thought to enable recruitment of Tudor domain-containing proteins (TDRDs), which might serve as platforms mediating interactions between various proteins in the piRNA pathway. We measured the binding affinity of the four individual extended Tudor domains (TDs) of murine TDRD1 protein for three different methylarginine-containing peptides from murine Piwi protein MILI. The results show a preference of TD2 and TD3 for consecutive MILI peptides, whereas TD4 and TD1 have, respectively, lower and very weak affinity for any peptide. The affinity of TD1 for methylarginine peptides can be restored by a single-point mutation back to the consensus aromatic cage sequence. These observations were confirmed by pull-down experiments with endogenous Piwi and Piwi-associated proteins. The crystal structure of TD3 bound to a methylated MILI peptide shows an unexpected orientation of the bound peptide, with additional contacts of nonmethylated residues being made outside of the aromatic cage, consistent with solution NMR titration experiments. Finally, the molecular envelope of the four tandem Tudor domains of TDRD1, derived from small angle scattering data, reveals a flexible, elongated shape for the protein. Overall, the results show that TDRD1 can accommodate different peptides from different proteins, and can therefore act as a scaffold protein for complex assembly in the piRNA pathway.

Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins

Arginine dimethylation plays critical roles in the assembly of ribonucleoprotein complexes in pre-mRNA splicing and piRNA pathways. We report solution structures of SMN and SPF30 Tudor domains bound to symmetric and asymmetric dimethylated arginine (DMA) that is inherent in the RNP complexes. An aromatic cage in the Tudor domain mediates dimethylarginine recognition by electrostatic stabilization through cation-π interactions. Distinct from extended Tudor domains, dimethylarginine binding by the SMN and SPF30 Tudor domains is independent of proximal residues in the ligand. Yet, enhanced micromolar affinities are obtained by external cooperativity when multiple methylation marks are presented in arginine- and glycine-rich peptide ligands. A hydrogen bond network in the SMN Tudor domain, including Glu134 and a tyrosine hydroxyl of the aromatic cage, enhances cation-π interactions and is impaired by a mutation causing an E134K substitution associated with spinal muscular atrophy. Our structural analysis enables the design of an optimized binding pocket and the prediction of DMA binding properties of Tudor domains.

A systematic analysis ofDrosophilaTUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors

PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) form the core of a gonad-specific small RNA silencing pathway that protects the animal genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology as TUDOR domains of various proteins bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analysed the Drosophila TUDOR protein family and identified four previously not characterized TUDOR domain-containing proteins (CG4771, CG14303, CG11133 and CG31755) as essential piRNA pathway factors. We characterized CG4771 (Vreteno) in detail and demonstrate a critical role for this protein in primary piRNA biogenesis. Vreteno physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Vreteno also interacts with the Tdrd12 orthologues CG11133 (Brother of Yb) and CG31755 (Sister of Yb), which are essential for the primary piRNA pathway in the germline and probably replace the function of the related but soma-specific factor Yb.

Structural basis for recognition of arginine methylated Piwi proteins by the extended Tudor domain

Arginine methylation modulates diverse cellular processes and represents a molecular signature of germ-line-specific Piwi family proteins. A subset of Tudor domains recognize arginine methylation modifications, but the binding mechanism has been lacking. Here we establish that, like other germ-line Tudor proteins, the ancestral staphylococcal nuclease domain-containing 1 (SND1) polypeptide is expressed and associates with PIWIL1/Miwi in germ cells. We find that human SND1 binds PIWIL1 in an arginine methylation-dependent manner with a preference for symmetrically dimethylated arginine. The entire Tudor domain and a bifurcated SN domain are required for this binding activity, whereas the canonical Tudor domain alone is insufficient for methylarginine ligand binding. Crystal structures show that the intact SND1 extended Tudor domain forms a wide and negatively charged binding groove, which can accommodate distinct symmetrically dimethylated arginine peptides from PIWIL1 in different orientations. This analysis explains how SND1 preferentially recognizes symmetrical dimethylarginine via an aromatic cage and conserved hydrogen bonds, and provides a general paradigm for the binding mechanisms of methylarginine-containing peptides by extended Tudor domains.

Structural basis for methylarginine-dependent recognition of Aubergine by Tudor

Piwi proteins are modified by symmetric dimethylation of arginine (sDMA), and the methylarginine-dependent interaction with Tudor domain proteins is critical for their functions in germline development. Cocrystal structures of an extended Tudor domain (eTud) of Drosophila Tudor with methylated peptides of Aubergine, a Piwi family protein, reveal that sDMA is recognized by an asparagine-gated aromatic cage. Furthermore, the unexpected Tudor-SN/p100 fold of eTud is important for sensing the position of sDMA. The structural information provides mechanistic insights into sDMA-dependent Piwi-Tudor interaction, and the recognition of sDMA by Tudor domains in general.

Structure and Ligand Binding of the Extended Tudor Domain of D. melanogaster Tudor-SN

The Tudor-SN protein (p100, SND1) has been implicated in a variety of cellular processes, such as transcription, processing of edited double-stranded RNA, and splicing regulation. Molecular details of these functions are not yet understood. Tudor domains have previously been shown to bind methylated ligands, such as methylated lysines and arginines. It has been suggested that the role of Tudor-SN in splicing may involve binding to such methylated ligands or to the methylated 5′ cap of spliceosomal snRNAs.Here, we report the crystal structure of the extended Tudor domain of Tudor-SN from Drosophila melanogaster to a resolution of 2.1 Å. NMR secondary chemical shifts, relaxation data, and residual dipolar couplings indicate that the solution and crystal structures are similar. Binding of various ligands was investigated by NMR. Binding sites and affinities were characterized by chemical shift perturbations. We show that the aromatic cage of the Tudor domain specifically binds a peptide containing symmetrically dimethylated arginines (sDMA) with micromolar affinity, while the same peptide comprising nonmethylated arginines does not show significant chemical shift perturbations. Tudor-SN preferentially recognizes sDMA over asymmetrically dimethylated arginine (aDMA). In contrast, two 5′ cap analogues with different methylation patterns, as well as mono-, di-, and trimethyllysines, show no binding.Our data demonstrate that the Tudor domain of Tudor-SN specifically recognizes sDMA-containing ligands. The aromatic cage of Tudor-SN is very similar to the one in the Tudor domain of the survival of motor neuron protein, which also recognizes sDMA peptides, indicating a conserved binding motif for this methylation mark. Recognition of sDMA in the C-terminal tails of spliceosomal Sm proteins suggests how Tudor-SN may interact with small nuclear ribonucleoprotein particles during the regulation of splicing.