Conserved and Widespread Expression of piRNA-Like Molecules and PIWI-Like Genes Reveal Dual Functions of Transposon Silencing and Gene Regulation in Pinctada fucata (Mollusca)

Songqian Huang,Shuichi Asakawa,Kiyohito Nagai,Shigeharu Kinoshita,Kazutoshi Yoshitake,Yuki Ichikawa,Fumito Omori,Kaoru Maeyama,Shugo Watabe,Md Asaduzzaman

doi:10.3389/fmars.2021.730556

Abstract

PIWI proteins and PIWI-interacting RNAs (piRNAs) suppress transposon activity in animals, thus safeguarding the genome from detrimental insertion mutagenesis. Recent studies revealed additional targets and functions of piRNAs in various animals. piRNAs are ubiquitously expressed in somatic tissues of the pearl oyster Pinctada fucata, however, the role of somatic piRNAs has not well characterized. This study reports the PIWI/piRNA pathway, including piRNA biogenesis and piRNA-mediated transposon silencing, and gene regulation in P. fucata. The biogenesis factors of PIWI, Zucchini, and HEN1, which are ubiquitous in somatic and gonadal tissues, were first identified in P. fucata using transcriptome analysis. Bioinformatics analyses suggested that different populations of piRNAs participate in the ping-pong amplification loop in a tissue-specific manner. In addition, a total of 69 piRNA clusters were identified in the genome of P. fucata based on the expression of piRNAs, which contained 26% transposons and enhanced for DNA/Crypton, LINE/CR1, SINE/Deu, and DNA/Academ. The expression patterns of the piRNAs and piRNA clusters in somatic tissues were not substantially different, but varied significantly between the somatic and gonadal tissues. Furthermore, locked-nucleic-acid modified oligonucleotide (LNA-antagonist) was used to silence single piRNA (piRNA0001) expression in P. fucata. Hundreds of endogenous genes were differentially expressed after piRNA silencing in P. fucata. Target prediction showed that some endogenous genes were targeted by piRNA0001, including twelve upregulated and nine downregulated genes after piRNA0001 silencing. The results indicated that piRNAs from somatic tissues may be related to gene regulation, whereas piRNAs from gonadal tissues are more closely associated to transposon silencing. This study will enhance our understanding of the role of piRNAs in mollusks, transposon silencing, and the regulatory function of the PIWI/piRNA pathway on protein-coding genes outside of germ line cells in P. fucata.

Highlights

Analysis of the PIWI/PIWI-interacting RNAs (piRNAs) pathway representative of several animals revealed an extensive diversity of lineage-specific adaptations, challenging the universal validity of data obtained from model organisms
PIWI proteins are characterized by two protein domains, namely the PAZ domain, an RNA-binding motif that binds the 3 end of short RNAs, and the PIWI domain, which is structurally similar to the RNaseH catalytic domain (Parker and Barford, 2006)
Numerous PIWI homologs have been identified in various organisms, such as four homologous Hiwi, Hili, Hiwi2, and Hiwi3 have been identified in Homo sapiens, and play a crucial role in human cancer and male germline cell development (Qiao et al, 2002; Sasaki et al, 2003; Liu et al, 2006); homologous Miwi, Mili, and Miwi2 were identified in Mus musculus, and their knockdown led to male sterility (Deng and Lin, 2002; Aravin et al, 2006; Lau et al, 2006); and two homolog, Ziwi and Zili, were identified in Danio rerio, and were both were crucial for germ cell differentiation and meiosis (Houwing et al, 2007, 2008)

Summary

Introduction

Animal species express three types of endogenous silencinginstigating small RNAs: microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs), and PIWI-interacting RNAs (piRNAs), based on their biogenesis mechanism and type of Argonaute-binding partners (Kim et al, 2009). miRNAs and endo-siRNAs, which are usually 20–23 nucleotides (nt) in length, are generated from double-stranded precursors by Dicer (Peters and Meister, 2007; Kim et al, 2009), whereas piRNAs are generated from single-stranded precursors independent of RNase III enzymes (Houwing et al, 2007), which are necessary for miRNA and endosiRNA biogenesis. piRNAs are associated with PIWI subfamily members of the Argonaute family of proteins, whereas miRNAs and endo-siRNAs are associated with AGO subfamily members. Animal species express three types of endogenous silencinginstigating small RNAs: microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs), and PIWI-interacting RNAs (piRNAs), based on their biogenesis mechanism and type of Argonaute-binding partners (Kim et al, 2009). PiRNAs are associated with PIWI subfamily members of the Argonaute family of proteins, whereas miRNAs and endo-siRNAs are associated with AGO subfamily members. The mechanisms underlying piRNA biogenesis and function remain largely unknown, mainly because the process has few similarities with the miRNA and endo-siRNA pathways. A comprehensive computational analysis of piRNA populations generated two models for piRNA biogenesis in various animals: the primary biogenesis pathway and the amplification loop or ping-pong cycle (Ishizu et al, 2012; Ross et al, 2014). Despite the recent characterization of several factors in the piRNA biogenesis pathway, it remains poorly understood

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