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

Chen Chen, Jing Jin, D. Andrew James, Melanie A. Adams-Cioaba, Jin Gyoon Park, Yahong Guo, Enrico Tenaglia, Chao Xu, Gerald Gish, Jinrong Min, Tony Pawson*

*Corresponding author for this work

Research output: Contribution to journalArticle

107 Citations (Scopus)

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,wehave 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.

Original languageEnglish (US)
Pages (from-to)20336-20341
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume106
Issue number48
DOIs
StatePublished - Dec 1 2009

Fingerprint

Arginine
Proteins
Germ Cells
Ligands
Small Interfering RNA
Tudor Domain
Immunoprecipitation
Protein Binding
Mutagenesis
Proteomics
Methylation
Drosophila
Cell Differentiation
Mass Spectrometry
Cytoplasm

ASJC Scopus subject areas

  • General

Cite this

Chen, Chen ; Jin, Jing ; James, D. Andrew ; Adams-Cioaba, Melanie A. ; Park, Jin Gyoon ; Guo, Yahong ; Tenaglia, Enrico ; Xu, Chao ; Gish, Gerald ; Min, Jinrong ; Pawson, Tony. / Mouse Piwi interactome identifies binding mechanism of Tdrkh Tudor domain to arginine methylated Miwi. In: Proceedings of the National Academy of Sciences of the United States of America. 2009 ; Vol. 106, No. 48. pp. 20336-20341.
@article{74ea11237189451a9721397a3f411c8d,
title = "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,wehave 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.",
author = "Chen Chen and Jing Jin and James, {D. Andrew} and Adams-Cioaba, {Melanie A.} and Park, {Jin Gyoon} and Yahong Guo and Enrico Tenaglia and Chao Xu and Gerald Gish and Jinrong Min and Tony Pawson",
year = "2009",
month = "12",
day = "1",
doi = "10.1073/pnas.0911640106",
language = "English (US)",
volume = "106",
pages = "20336--20341",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "48",

}

Mouse Piwi interactome identifies binding mechanism of Tdrkh Tudor domain to arginine methylated Miwi. / Chen, Chen; Jin, Jing; James, D. Andrew; Adams-Cioaba, Melanie A.; Park, Jin Gyoon; Guo, Yahong; Tenaglia, Enrico; Xu, Chao; Gish, Gerald; Min, Jinrong; Pawson, Tony.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, No. 48, 01.12.2009, p. 20336-20341.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mouse Piwi interactome identifies binding mechanism of Tdrkh Tudor domain to arginine methylated Miwi

AU - Chen, Chen

AU - Jin, Jing

AU - James, D. Andrew

AU - Adams-Cioaba, Melanie A.

AU - Park, Jin Gyoon

AU - Guo, Yahong

AU - Tenaglia, Enrico

AU - Xu, Chao

AU - Gish, Gerald

AU - Min, Jinrong

AU - Pawson, Tony

PY - 2009/12/1

Y1 - 2009/12/1

N2 - 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,wehave 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.

AB - 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,wehave 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.

UR - http://www.scopus.com/inward/record.url?scp=73949084329&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=73949084329&partnerID=8YFLogxK

U2 - 10.1073/pnas.0911640106

DO - 10.1073/pnas.0911640106

M3 - Article

VL - 106

SP - 20336

EP - 20341

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 48

ER -