Mouse Piwi interactome identifies binding mechanism of Tdrkh Tudor domain to arginine methylated Miwi Chen Chena, Jing Jina, D. Andrew Jamesa, Melanie A. Adams-Cioabab, Jin Gyoon Parka, Yahong Guob, Enrico Tenagliaa,c, Chao Xub, Gerald Gisha, Jinrong Minb,d, and Tony Pawsona,c,1 aSamuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; bStructural Genomics Consortium, University of Toronto, Toronto, ON, Canada M5G 1L6; and cDepartment of Molecular Genetics and dDepartment of Physiology, University of Toronto, Toronto, ON, Canada M5S 1A8 Contributed by Tony Pawson, October 8, 2009 (sent for review September 15, 2009) Tudor domains are protein modules that mediate protein–protein spermatogenesis, and small RNA pathways (7–9). However, the interactions, potentially by binding to methylated ligands. A group of binding properties of these germline Tudor proteins are poorly germline specific single and multiTudor domain containing proteins understood. (TDRDs) represented by drosophila Tudor and its mammalian or- Piwi proteins are conserved germline-specific Argonaute fam- thologs Tdrd1, Tdrd4/RNF17, and Tdrd6 play evolutionarily conserved ily members that are associated with Piwi-interacting RNAs roles in germinal granule/nuage formation and germ cell specification (piRNAs), and thereby function in piRNA-mediated posttranscrip- and differentiation. However, their physiological ligands, and the tional silencing (10). Three murine Piwi paralogs, Miwi, Mili, and biochemical and structural basis for ligand recognition, are largely Miwi2, play pivotal roles in germ cell development, transposon unclear. Here, by immunoprecipitation of endogenous murine Piwi silencing and spermatogenesis (11–13). The presence of multiple proteins (Miwi and Mili) and proteomic analysis of complexes related arginine-glycine and arginine-alanine (RG/RA)-rich clusters at the to the piRNA pathway, we show that the TDRD group of Tudor N-termini of these proteins prompted us to question whether these proteins are physiological binding partners of Piwi family proteins. In RG/RA motifs can be methylated in vivo and thereby serve as addition, mass spectrometry indicates that arginine residues in RG docking sites for the binding of various germline Tudor proteins. repeats at the N-termini of Miwi and Mili are methylated in vivo. To test this hypothesis, we performed a comprehensive pro- Notably, we found that Tdrkh/Tdrd2, a novel single Tudor domain teomic analysis of Miwi and Mili complexes in adult male germ cells CELL BIOLOGY containing protein identified in the Miwi complex, is expressed in the and determined the methylation status of these Piwi proteins. We cytoplasm of male germ cells and directly associates with Miwi. show that several germline Tudor proteins are physiological binding Mutagenesis studies mapped the Miwi–Tdrkh interaction to the very partners of the Piwi family. In particular, we identify Tdrkh as a N-terminal RG/RA repeats of Miwi and showed that the Tdrkh Tudor novel Miwi-interacting protein that binds Miwi through its single domain is critical for binding. Furthermore, we have solved the crystal Tudor domain, likely via arginine methylation, as suggested by a structure of the Tdrkh Tudor domain, which revealed an aromatic combination of mass spectrometry, mutagenesis, and structural binding pocket and negatively charged binding surface appropriate analysis. for accommodating methylated arginine. Our findings identify a methylation-directed protein interaction mechanism in germ cells Results mediated by germline Tudor domains and methylated Piwi family Tudor Domain-Containing Proteins Are Major Physiological Binding proteins, and suggest a complex mode of regulating the organization Partners of Piwi Family Proteins. To test whether Tudor domain and function of Piwi proteins in piRNA silencing pathways. family proteins comprise the in vivo binding partners of the Piwi proteins, we immunoprecipitated endogenous Miwi and Mili from udor domains, together with Chromo, MBT, PWWP, and lysates of adult testes and purified the complexes by acid elution. To TAgenet-like domains, comprise the ‘‘Tudor Royal Family’’ of obtain a comprehensive survey of the components of the Piwi domains (1). The core structure of this protein domain superfamily complexes, we used a gel-free liquid chromatography coupled is characterized by an antiparallel ␤-barrel-like topology and me- tandem mass spectrometry (LC-MS/MS) approach employing solid diates protein–protein interactions, in some cases by recognizing phase tryptic digestion. This technique allowed us to unambigu- methylated lysine/arginine-containing ligands with a binding site ously identify an extensive list of candidate proteins that specifically composed of aromatic residues (2, 3). Their methylated target associated with Piwi proteins (Fig. 1). Hierarchical clustering of 2 proteins are implicated in diverse biological processes such as independent repeats of Miwi and Mili immunoprecipitations (IP) chromatin remodeling and RNA splicing. For example, the Tudor with their respective IgG control IPs reproducibly revealed distinct domain of Smn binds to methylated arginine-glycine (RG) motifs protein complex profiles for Miwi and Mili (Fig. 1). We found on Sm proteins essential for spliceosome assembly (4), while the proteins that were specifically associated with either Miwi (Fig. 1A, Tudor domains of Jmjd2a bind to methylated lysines in histone blue box and Fig. S2) or Mili (Fig. 1A, red box), and proteins shared H4K20 (5). by both Miwi and Mili complexes (Fig. 1A, cyan box). All of these Drosophila Tudor, the founding member of the Tudor domain proteins were absent from the IgG control IPs, which contain family, is a germ cell-specific protein with multiple Tudor domains nonspecific binding proteins (Fig. S2). In this analysis, we observed and is involved in germ plasm formation and germ cell specification several previously known Piwi-interacting proteins or piRNA path- (6). By analyzing the expression pattern of mammalian genes encoding Tudor domain proteins, we identified a group whose Author contributions: C.C., J.J., and T.P. designed research; C.C., J.J., D.A.J., M.A.A.-C., Y.G., expression is highly enriched in germ cells, which we therefore term E.T., C.X., and G.G. performed research; C.C., J.G.P., and J.M. analyzed data; and C.C. and germline Tudor proteins (Tdrd1, Tdrkh/Tdrd2, RNF17/Tdrd4, T.P. wrote the paper. Tdrd5, Tdrd6, Tdrd7, Stk31/Tdrd8, Tdrd9, Tdrd10, Akap1) (sup- The authors declare no conflict of interest. porting information (SI) Fig. S1). While the physiological functions Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, of germline proteins with a single Tudor domain (Tdrkh, Tdrd5, www.pdb.org (PDB ID code 3fdr). Stk31, and Tdrd9) are largely unknown, mouse knockout studies of 1To whom correspondence should be addressed. E-mail: [email protected]. Tdrd1, Tdrd4, and Tdrd6 have revealed crucial roles for these This article contains supporting information online at www.pnas.org/cgi/content/full/ multiTudor domain proteins in nuage/chromatoid body formation, 0911640106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0911640106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 29, 2021 AB Ddx1 Kctd3 Pacsin1 1700020L24Rik Igf2r Golga5 Sucla2 Miwi_IP_2 Mili_IP_2 IgG_IP_(Mili1) IgG_IP_(Mili2) Mili_IP_1 Miwi_IP_1 IgG_IP_(Miwi 1) IgG_IP_(Miwi 2) Trpd52l3 1110012J17Rik 100046745 Kng1 Cep72 1700020L24Rik Ppp2r1a Pacsin2 Cul9 Pacsin2 Ddx1 Ilf3 Psmd2 Sccpdh Tdrd7 OTTMUSG00000023442 8030462N17Rik Stk22s1 Rai14 Rnf213 C330043M08Rik Cep72 Tdrd6 Git1 Stk31 ENSMUSG00000057808 Atp9a ENSMUSG00000076684 Hnrnpl Spata20 8030462N17Rik Stk22s1 Tpr Hdac6 Rai14 Sept2 Rab3ip B230339M05Rik Hsp90ab1 Tjp1 Birc6 Sept9 ENSMUSG00000076684 Ccdc6 Tdrkh Stk31 Kif17 Tjp1 Ccdc6 Golga5 Birc6 Kif17 Pgp Hsp90b1 Ssb Rnf213 Rpl5 1110012J17Rik Acta1 Tpr Rps4y2 Isyna1 Eef1g Hnrnpf Rps9 Hsp90ab1 Lta4h Igf2r Ilf3 Ppp2r1a Akap4 Tdrd6 Amotl1 Atp9a Phldb1 Spata20 Kctd3 Myst3 Pacsin1 Trpd52l3 Sept9 Phldb1 Pkd1l1 Hnrnpl OTTMUSG00000023442 Vps13d Miwi Ybx2 Ahsg ENSMUSG00000076502 C330043M08Rik Fcgr4 Krt5 Psmd2 Hdac6 Rps9 Sept7 Eef1a1 Tdrkh C3 Igh-VX24 Pcca LOC100046359 ENSMUSG00000076718 Hsp90aa1 Igh-6 Dbt Hpx Amotl1 ENSMUSG00000076683 LOC675759 Git1 ENSMUSG00000076939 Igh-VJ558 Fgb Sept2 Hrg Gc Igk-C ENSMUSG00000076709 Pkd1l1 Pzp Ddx4 Aldh1a1 Fgg Sccpdh Mug1 Myst3 Tubb3 Eef1g OTTMUSG00000015054 Eef2 ENSMUSG00000076665 Rpl5 Atp5b ENSMUSP00000112082 Keap1 Ldhc Ttn Dhx9 B230339M05Rik Khsrp Akap4 Trim21 Myo18a Rassf2 Ryr1 Acaa2 Srrm2 Nasp Wdr37 Psmc3 Tns1 Fxr1 ENSMUSG00000057808 Golga3 Nucb1 Ddx5 Tln1 Vim Rps7 1700012A16Rik Sucla2 Ruvbl2 Pbxip1 Hnrnpf Cct8 Hspa8 Tdrd7 Cct2 Dync1h1 Acta1 Rpl23 Cct5 Crybg3 Rab3ip Ddx4 Cct6a Cul9 Trim28 Dnpep Fth1 Hnrnpc Mtap4 Pcbp1 Hspa4l 4932438A13Rik Suclg1 Snrpd2 Tdrd1 Rps13 Rpl28 Rps14 ENSMUSG00000074479 Prdx1 C1qa Cct4 Slc25a31 Tcp1 Hnrnpu ENSMUSG00000076557 Mov10l1 ENSMUSG00000076652 LOC100041230 Blzf1 ENSMUSG00000076692 ENSMUSG00000076691 Rpl8 ENSMUSG00000073028 ENSMUSG00000076649 ENSMUSG00000076695 ENSMUSG00000076736 LOC630322 Fip1l1 ENSMUSG00000076740 Hnrnpm Matr3 Hspa9 Vcp Fga Rps5 Hnrnpa1 Hadha Mocs1 Trf Ywhaz Rps3 Vasp Cct3 Rps20 Rps25 Rpl18 Rps18 Napa Rps26 Mili 1700019E19Rik Zfp219 Fip1l1 Fyttd1 Enthd1 Gramd3 Gbf1 Snrpn Atp5a1 4932438A13Rik Fyttd1 Caprin1 G3bp1 Vasp Nucb1 Tns1 Sorbs2 Blzf1 Cul3 Eml4 Txndc2 Sssca1 Mov10l1 Txndc2 Sssca1 Enthd1 Mocs1 Snrpd2 Crybg3 G3bp1 Pbxip1 Napa 1700019E19Rik Wdhd1 Gramd1a Gas2l1