Uniting germline and stem cells: the function of Piwi proteins and the piRNA pathway in diverse organisms

Abstract

The topipotency of the germline is the full manifestation of the pluri- and multipotency of embryonic and adult stem cells, thus the germline and stem cells must share common mechanisms that guarantee their multipotentials in development. One of the few such known shared mechanisms is represented by Piwi proteins, which constitute one of the two subfamilies of the Argonaute protein family. Piwi proteins bind to Piwi-interacting RNAs (piRNAs) that are generally 26 to 31 nucleotides in length. Both Piwi proteins and piRNAs are most abundantly expressed in the germline. Moreover, Piwi proteins are expressed broadly in certain types of somatic stem/progenitor cells and other somatic cells across animal phylogeny. Recent studies indicate that the Piwi-piRNA pathway mediates epigenetic programming and posttranscriptional regulation, which may be responsible for its function in germline specification, gametogenesis, stem cell maintenance, transposon silencing, and genome integrity in diverse organisms.

Figure 1. Molecular functions of Piwi proteins

(A) In Drosophila, Piwi is a nuclear protein that binds Heterochromatin Protein 1a (HP1a) and is required for H3K9 (Lysine 9 of Histone 3) methylation and heterochromatin formation (10, 84). (B) In the mouse, Miwi2 protein is enriched in the nucleus and is required for de novo DNA methylation in male germ cells (4). (C) Piwi proteins in the nuage participate in a ping-pong mechanism that simultaneously produces new piRNAs and degrades active transposon mRNAs, see Figure 2 for details (9, 41). (D) The Drosophila Piwi proteins Aub and Ago3 are required for the deadenylation and degradation of maternal mRNAs during the maternal-to-zygotic transition (101). (E) In Drosophila, mouse, and Xenopus, 10–35% of piRNAs are processed from the 3′UTR of select mRNAs. This may represent a mechanism for regulating these mRNAs in cis. In addition, the resulting piRNAs may regulate gene expression in trans (100). (F) Mili and Miwi associate with polysomes in the mouse male germline to positively regulate gene expression (40, 117). (G) In Drosophila, Hsp90 and Hop form a complex with Piwi, which leads to Piwi phosphorylation and is required for Piwi-dependent canalization in the nucleus (36).

Figure 2. The Ping Pong model of piRNA biogenesis and transposon repression in Drosophila (9, 41)

Piwi proteins generate the 5′ ends of new piRNAs that are derived from either piRNA cluster transcripts (primary piRNAs) or active mRNA transposons (secondary piRNAs). Piwi- and Aub-bound primary piRNAs are either maternally loaded or processed by the primary pathway. Next, Piwi and Aub, guided by their bound antisense piRNAs, bind to active transposon mRNAs and create the new 5′ ends of sense secondary piRNAs. Cleavage of the transposon mRNA occurs 10 nucleotides upstream of the adenine that is complimentary to the 5′uridine of the primary piRNA. Ago3 proteins, bound to secondary piRNAs, are then capable of producing the 5′ end of new primary piRNAs made from piRNA cluster transcripts. It is not known how the 3′ end of piRNAs are created in the ping pong mechanism.

Figure 3. Piwi proteins are conserved in metazoans

(A) A metazoan phylogenetic tree. Bilatarians are classified as either protostomes (green) or deuterostomes (red) based on differences in embryo development. Non-bilatarian phyla (blue) include cnidarians, ctenophores, and sponges. Tetrahymena is a ciliated protist that also expresses Piwi proteins. (B) Phylogenetic analysis of Argonaute proteins demonstrates a clear distinction between the Piwi (yellow) and Argonuate (purple) subfamilies. Phylogram generated using Phylogeny.fr (29). Numbers on the tree demonstrate branch supports. NCBI accession numbers from top to bottom are as follows: Prg-1 (NM_059720), Prg-2 (NM_068593), Piwi (NM_057527), Aubergine (NM_001103674), Ago3 (NM_001043162), Smedwi-2 (EU586258), Zili (NM001080199), Mili (NM_021308), Hili (NM_001135721), EfPiwiA (AB533505), BpPiwi1 (AB455103), Miwi2 (NM_177905), Hiwi2 (NM_152431), Ziwi (NM_18338), Miwi (NM_021311), Hiwi (NM_004764.4), ClytiaPiwi (EU199802), EfPiwiB (AB533506.1), Smedwi1 (DQ186985), Smedwi2 (DQ186986), Twi11 (EU183125), Twi1 (AB084111), EfAgoA (AB533507), Drosophila Ago1 (NM_166020), Zebrafish Ago2 (XM_694134), Mouse Ago2 (AY135688), Human Ago2 (NM_001164623), Mouse Ago3 (AY135689), Zebrafish Ago3 (XM_690923), Mouse Ago4 (AY135690), Mouse Ago1 (AY135687), Drosophila Ago2 (NM_140518).