Biology of PIWI-interacting RNAs: new insights into biogenesis and function inside and outside of germlines

Abstract

PIWI-interacting RNAs (piRNAs) are endogenous small noncoding RNAs that act as guardians of the genome, protecting it from invasive transposable elements in the germline. Animals lacking piRNA functions show defects in gametogenesis and exhibit sterility. Their descendants are also predisposed to inheriting mutations. Thus, the piRNA pathway has evolved to repress transposons post-transcriptionally and/or transcriptionally. A growing number of studies on piRNAs have investigated piRNA-mediated gene silencing, including piRNA biogenesis. However, piRNAs remain the most enigmatic among all of the silencing-inducing small RNAs because of their complexity and uniqueness. Although piRNAs have been previously suggested to be germline-specific, recent studies have shown that piRNAs also play crucial roles in nongonadal cells. Furthermore, piRNAs have also recently been shown to have roles in multigenerational epigenetic phenomena in worms. The purpose of this review is to highlight new piRNA factors and novel insights in the piRNA world.

Figure 1.

piRNA biogenesis in Drosophila and mice consists of the primary piRNA processing pathway and the amplification loop. The left side shows a model of the primary processing pathway in flies. The primary transcripts of piRNA clusters are shortened into piRNA-like small RNAs. The factors necessary for this process largely remain to be determined. Recent studies have suggested that Zucchini might be an endonuclease forming the 5′ ends of piRNAs. The mouse Zucchini homolog (MitoPLD) might also function as a nuclease to produce primary piRNAs. piRNA-like small RNAs are loaded onto PIWI proteins (shown in pink) and then trimmed from the 3′ end to the size of mature piRNAs by an unknown nuclease. Hsp83/Shu may play a role in the Piwi loading step. piRNAs are 2′-O-methylated by HEN1/Pimet. In flies, the PIWI proteins that associate with primary piRNAs are Piwi (green) and Aub (blue). Piwi associated with piRNAs is translocated to the nucleus and thus likely does not contribute to the amplification loop. Aub associated with piRNAs now triggers the Ping-Pong cycle by cleaving transposon transcripts. Ago3 (orange) loaded with secondary piRNAs in turn produces piRNAs that associate with Aub. Transposon transcripts are cleaved through this amplification cycle, resulting in cytoplasmic transposon silencing. In mice, primary piRNAs are loaded onto MILI (yellow) and MIWI (red). MIWI associated with pachytene piRNAs functions in cytoplasmic silencing. The targets remain largely unknown. MILI associated with primary piRNAs contributes to the Ping-Pong cycle to produce piRNAs that associate with MIWI2 (purple), upon which MIWI2 is localized to the nucleus to accomplish nuclear silencing. The contribution of MIWI2 to the Ping-Pong cycle may be negligible. MILI might operate a homotypic Ping-Pong cycle as indicated. HSP90/FKBP6 plays a role in producing secondary piRNAs that associate with MIWI2. HEN1/Pimet 2′-O-methylates secondary piRNAs, the products of the Ping-Pong cycle.

Figure 2.

piRNA biogenesis factors in Drosophila. Drosophila piRNA factors can be subgrouped into PIWI proteins, TDRD proteins, and others. The cellular localizations in ovaries and protein–protein interaction partners of individual factors are summarized on the right. (N.D.) Not determined. Interaction of Tral, Me31B, and TER94 with Papi was reported by Liu et al. (2011). HP1 interaction with Qin/Kumo was reported by Anand and Kai (2011). In addition to perinuclear localization in the nurse cells, Kumo also appears as foci in the nuclei of the germ cells in the germarium (indicated by an asterisk). Interaction of MTOC proteins with Mael was reported by Sato et al. (2011).

Figure 3.

Zucchini in primary piRNA biogenesis in Drosophila ovarian somas. (A) Long, single-stranded piRNA precursors are transcribed from piRNA clusters in the genome and are processed into piRNA intermediates through an unknown mechanism. (B) An enlarged cartoon of the area shown by a dotted square in A: The RNA helicase Armi and the Tud domain-containing RNA helicase Yb localize to Yb bodies, which are often located adjacent to mitochondria. Armi and Yb form a complex that contains piRNA intermediates with 5′-hydroxyl and 3′-cyclic phosphate ends. Nascent, piRNA-free Piwi transiently localizes to Yb bodies to interact with the Armi–Yb complex. Zuc is anchored on the outer surfaces of mitochondria with the catalytic site facing the cytosol and processes piRNA intermediates into piRNA fragments, which are loaded onto Piwi. Primary piRNAs show a strong bias for 1-U, although Zuc endonuclease showed little sequence specificity in vitro. These findings lead to the possibility that cofactors may be involved in 1-U determination in vivo (“cofactors?”). Alternatively, Piwi may preferentially bind 1-U piRNAs among all Zuc cleavage products (“selection?”). Vret and Shu are newly characterized piRNA factors that localize to Yb-bodies. The functions of Vret and Shu have not been fully elucidated. The enzymes participating in 3′ end formation of piRNAs remain to be identified.

Figure 4.

piRNAs in C. elegans. C. elegans piRNAs are 21 nt in length with 1-U (21U-RNAs) and are loaded onto the PIWI protein PRG-1. piRNAs are genomically encoded in two large clusters on chromosome IV. These clusters are depleted of protein-coding genes. piRNAs have a characteristic sequence motif ∼42 nt upstream of the start of the small RNA, which may be required for RNA polymerase II (Pol II) transcription. piRNAs scan virtually all transcripts expressed in the germline and guide PRG-1 to targets by means of relaxed base-pairing with up to four mismatches. PRG-1–piRNA complexes then recruit RNA-dependent RNA polymerases (RdRPs) to the target site to produce 22G-RNAs (which are loaded onto WAGOs) that silence foreign genetic elements (Exo). WAGOs that localize to the cytoplasm mediate mRNA turnover (PTGS), whereas WAGOs that localize to the nucleus mediate transcriptional gene silencing (TGS). The CSR-1 22G RNA pathway may provide a memory of self (Endo) and act as an anti-silencing signal.