Beyond transposons: the epigenetic and somatic functions of the Piwi-piRNA mechanism

Piwi-interacting RNAs (piRNAs) were reported in 2006 as a novel class of small non-coding RNAs associated with Piwi proteins of the Argonaute/Piwi family. Recent studies have revealed not only the biogenesis of piRNAs and their roles in transposon silencing, but also the function of the Piwi-piRNA pathway in epigenetic and post-transcriptional regulation of gene expression. In addition, the function of this pathway in somatic cells has also been more systematically characterized. The new findings reveal the Piwi-piRNA pathway as a more general mechanism of gene regulation.

Introduction

Three major types of small RNAs, siRNAs, miRNAs, and piRNAs, associate with proteins in the Argonaute/Piwi family to execute sequence-specific gene silencing. Among the three types of small RNAs, piRNAs were discovered most recently and remain the least characterized. They are named by their exclusive association with Piwi but not Argonaute (Ago) subfamily proteins. Unlike the broad expression of siRNAs and miRNAs in most cell and tissue types, piRNAs are highly enriched in the germline. This expression pattern and the findings that mutations of piRNA biogenesis factors result in either germline stem cell loss or sterility of the mutant animals demonstrate the critical impact the Piwi-piRNA pathway exerts on germline development and functions [1, 2, 3, 4, 5, 6]. Strong hypormophic mutations of Drosophila piwi result in reduced germline stem cell division and eventual germline stem cell loss [3, 7]. Reduced level of Piwi homologs in Caenorhabditis elegans result in reduced germ cell division and reduced fertility [7, 8]. Knockout mutations of mouse Piwi proteins Mili, Miwi, and Miwi2 result in male sterility in all cases, spermatogonial stem cell arrest and eventual loss in Mili and Miwi2 mutants, and meiotic arrest in Miwi mutant [4, 5, 6, 9, 10]. Genetic analysis of Piwi functions in the germline of these model organisms reinforced the critical role of Piwi proteins in germline development.

Numerous excellent reviews have detailed piRNA biogenesis pathway and contrasted it with miRNA and siRNA biogenesis [11, 12, 13]. Here we highlight some less discussed points. First, in contrast to the ∼500 loci encoding miRNAs in most eukaryotic genomes, the number of piRNAs ranges up to 100 000's. Second, both the RNA polymerase transcribing the primary transcripts and the nuclease generating primary piRNAs from the primary transcripts are yet to be identified; an endonuclease Zucchini has been implicated in generating the 5′ end of piRNA [14, 15]. Third, the mechanism of RNA target selection by the piRNA-induced silencing complex is unclear. Fourth, the mechanism by which PIWI-piRNA complexes regulate epigenetic processes via sequence specificity remains unclear.

The questions highlighted above remain unsolved partially because studies of piRNA functions have mostly been focused on transposon silencing in the germline. Indeed, mutations of genes involved in piRNA biogenesis result in increased transposition and DNA damage in germ cells [16, 17] and because a subpopulation of piRNAs appears to suppress retrotransposon activities in the germline [18, 19]. These studies, however, often overlook substantial evidence demonstrating that the Piwi-piRNA pathway significantly influences epigenetic mechanisms. Drosophila Piwi and its homolog Aubergine have been shown to affect transcriptional silencing [20], to be required for heterochromatin formation [21], and to affect Polycomb Group-mediated transgene silencing [22]. Furthermore, molecular and biochemical analyses of Piwi and piRNAs demonstrated that Piwi protein directly associates with and recruits HP1a [23, 24], that the Piwi-piRNA complex directly binds to piRNA-complementary sequence in the genome and is required for proper histone modification of the sequence [25], and that maternally deposited piRNAs can epigenetically mediate transposon suppression in the offspring [26]. In addition, analyses of mouse Piwi homologs, Mili and Miwi2, indicated that they mediate DNA methylation in the male germline during embryogenesis [27, 28, 29]. Together, the above observations implicate epigenetic function of the Piwi-piRNA pathway and its importance for germline health and functions.