Functional analysis of Tudor-domain-containing proteins in the zebrafish germline

Abstract

The Argonaute protein family consists of two distinct sub-clades: AGO and PIWI proteins. AGO proteins can form RNA induced silencing complex (RISC) and are responsible for RNAi and miRNA pathways. PIWI proteins are mostly expressed in the germline and together with specific small RNA groups, named PIWI-interacting RNAs (piRNAs), they function in a germline-specific RNAi-like pathway. PIWI proteins are responsible for piRNA biogenesis and germline development/ function in many species. Zebrafish genome encodes two PIWI proteins: Ziwi and Zili. Loss of Ziwi or Zili function, leads to sterility and transposon de-repression in the germline. PIWI proteins in various species have been reported to interact with a special protein family, Tudor domain containing (Tdrd) proteins. Tdrd mutants often phenocopy piwi mutants, suggesting their roles in the PIWI-piRNA pathway. Tdrd proteins interact with proteins with methylated arginine residues in the RG motifs, which are also found in the N-terminus of PIWI proteins. Via reverse genetic screening, we obtained mutant zebrafish alleles in tdrd1, tdrd6 and tdrd9 genes. These three tdrd genes are germline-specific. We found that these three tdrd proteins are involved in the piRNA functions to different extent. Tdrd1 function as a scaffold to facilitate the piRNA pathway by recruiting PIWI proteins and their targets. Tdrd9 is important in maintaining the germ meiotic stem cells. Tdrd6 is a possible reservoir for Ziwi-specific piRNAs and assisting Ziwi targeting retrotransposons. Moreover, Tdrd6 is involved in germ plasm assembly in the early stage of embryogenesis.