Mouse Piwi interactome identifies binding mechanism of Tdrkh Tudor domain to arginine methylated Miwi

Abstract

Tudor domains are protein modules that mediate protein-protein interactions, potentially by binding to methylated ligands. A group of germline specific single and multiTudor domain containing proteins (TDRDs) represented by drosophila Tudor and its mammalian orthologs Tdrd1, Tdrd4/RNF17, and Tdrd6 play evolutionarily conserved roles in germinal granule/nuage formation and germ cell specification and differentiation. However, their physiological ligands, and the biochemical and structural basis for ligand recognition, are largely unclear. Here, by immunoprecipitation of endogenous murine Piwi proteins (Miwi and Mili) and proteomic analysis of complexes related to the piRNA pathway, we show that the TDRD group of Tudor proteins are physiological binding partners of Piwi family proteins. In addition, mass spectrometry indicates that arginine residues in RG repeats at the N-termini of Miwi and Mili are methylated in vivo. Notably, we found that Tdrkh/Tdrd2, a novel single Tudor domain containing protein identified in the Miwi complex, is expressed in the cytoplasm of male germ cells and directly associates with Miwi. Mutagenesis studies mapped the Miwi-Tdrkh interaction to the very N-terminal RG/RA repeats of Miwi and showed that the Tdrkh Tudor domain is critical for binding. Furthermore, we have solved the crystal structure of the Tdrkh Tudor domain, which revealed an aromatic binding pocket and negatively charged binding surface appropriate for accommodating methylated arginine. Our findings identify a methylation-directed protein interaction mechanism in germ cells mediated by germline Tudor domains and methylated Piwi family proteins, and suggest a complex mode of regulating the organization and function of Piwi proteins in piRNA silencing pathways.

Fig. 1.

Proteomic analysis of endogenous Piwi complexes identifies Tudor domain family proteins as physiological binding partners. (A) Hierarchical clustering of proteins identified by 2 independent immunoprecipitations (IP) of Miwi, Mili, and corresponding IgG controls through tandem mass spectrometry. Proteins for which 3 or more peptides were identified are shown. Proteins that interact specifically with Miwi or Mili are illustrated in blue and red boxes, respectively. The cyan box indicates common proteins associated with both Miwi and Mili. (B) Identification of multiple germline Tudor domain proteins in Miwi and Mili complexes. Miwi and Mili protein interaction networks are shown with baits in blue and Tudor domain proteins in orange. Proteins represented by 3 or more peptides and not present in the IgG control IP, and proteins represented by peptides with a 5-fold increase over the IgG control IP are shown.

Fig. 2.

Arginine methylation sites detected on endogenous Miwi and Mili by mass spectrometry. N-terminal RG/RA-rich sequences are show in red. Identified methylation sites (Me) are shown above the relevant arginine, with the residue numbers underneath.

Fig. 3.

Expression kinetics and cellular localization of Tdrkh in the mouse testis. (A) Tissue distribution of Tdrkh protein by Western blotting. Tissue lysates were probed with anti-Tdrkh antibody; anti-Tubulin antibody was used as a control. (B) Western blot analysis of temporal Tdrkh protein expression in mouse testes. ES: mouse embryonic stem cell, P: postnatal day. (C–F) Immunofluorescence staining of Tdrkh (green) in testes of different developmental ages. P: postnatal day. (G–I) Colocalization of Tdrkh and Mvh in the adult testis.

Fig. 4.

Tdrkh directly interacts with Miwi in vivo and in vitro through its Tudor domain. (A) Miwi is among the top specific interaction partners complexed with Tdrkh. Immunoprecipitation of Tdrkh from adult testis lysate and gel-free mass spectrometry were performed. Specific binding proteins are ranked based on the total peptide number identified. The top 5 Tdrkh interacting proteins with total peptide numbers and percentage of sequence coverage are shown for 2 independent immunoprecipitation experiments. (B) The interaction between Tdrkh and Miwi is RNA independent. Endogenous Tdrkh and Miwi were immunoprecipitated from adult testis lysates treated with or without RNaseA using anti-Tdrkh and anti-Miwi antibodies, respectively and immunoblotted with anti-Tdrkh antibody. (C) Tdrkh binds to the first cluster of RG/RA repeats on Miwi via its Tudor domain. HEK293T cells were cotransfected with Flag-Miwi or Flag-Miwi (R-K) mutants and GFP-Tdrkh or GFP-Tdrkh Tudor domain mutant (D390A, F391A). Flag-tagged protein complexes immunoprecipitated from cell extracts and whole cell lysates (WCL) were probed with anti-GFP and anti-Flag antibodies. The scheme of Miwi arginine mutations is shown in the bottom panel, with a red cross indicating an R-K mutant.

Fig. 5.

Crystal structure of the Tudor domain of Tdrkh. (A) Ribbon representation of the Tdrkh Tudor domain crystal structure. The residues comprising the aromatic binding pocket are shown in yellow. (B) Ribbon representation of the Snd1 Tudor domain crystal structure. (C) Surface representation of the Tdrkh Tudor domain crystal structure. (D) Surface representation of the Snd1 Tudor domain crystal structure. (E) Molecular docking of a GRG peptide with sDMA into the aromatic cage of the Tdrkh Tudor domain.