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.