piRNA processing within non-membrane structures is governed by constituent proteins and their functional motifs

Abstract

Discovered two decades ago, PIWI-interacting RNAs (piRNAs) are crucial for silencing transposable elements (TEs) in animal gonads, thereby protecting the germline genome from harmful transposition, and ensuring species continuity. Silencing of TEs is achieved through transcriptional and post-transcriptional suppression by piRNAs and the PIWI clade of Argonaute proteins within non-membrane structured organelle. These structures are composed of proteins involved in piRNA processing, including PIWIs and other proteins by distinct functional motifs such as the Tudor domain, LOTUS, and intrinsic disordered regions (IDRs). This review highlights recent advances in understanding the roles of these conserved proteins and structural motifs in piRNA biogenesis. We explore the molecular mechanisms of piRNA biogenesis, with a primary focus on Drosophila as a model organism, identifying common themes and species-specific variations. Additionally, we extend the discussion to the roles of these components in nongonadal tissues.

Keywords: Drosophila germline; Tudor domain‐containing proteins; liquid–liquid phase separation; non‐membrane nuage; piRNAs.

Fig. 1

Conservation of ping‐pong and phasing pathways along with proteins involved in these pathways across metazoans. The presence or absence of the ping‐pong and phasing pathways, along with associated proteins Zucchini/MitoPLD, PARN, PNLDC1, and Nibbler/Mut‐7, are shown. ‘Ticks’, ‘NO’ and ‘–’ denote the presence of the protein, the absence of the protein, and unavailability of the data, respectively. The numbers in the PIWI column indicate the number of PIWI proteins in each species. Evolutionary relationships among animal phyla are presented in a phylogenetic tree; however, the branch lengths do not represent evolutionary distances. Taxonomic groups mentioned in the text are highlighted in red.

Fig. 2

Transcription and processing machinery for piRNA Precursors in Drosophila melanogaster . (A) piRNA biogenesis in germline cells. Rhino recognizes H3K9me3 modifications on dual‐strand piRNA clusters and forms the RDC complex with Deadlock and Cutoff. This complex facilitates promoter‐independent transcription and contributes to the nuclear export of piRNA precursor transcripts via Bootlegger, which recruits the Nxf3–Nxt1 complex. Nxf3 together with exportin Crm1, then transports the piRNA precursors to the perinuclear nuage on the cytoplasmic side. Within nuage, Aub and Ago3 sequentially cleave TE mRNAs and cluster transcripts, respectively, in turns in the ping‐pong cycle. Nibbler trims the 5′ ends of pre‐piRNAs, while Hen1 2′‐O‐methylates their 3′ ends, resulting in mature piRNAs. piRNA precursors can also be processed through phasing. Aub‐bound piRNA precursors are transported to the mitochondrial outer membrane by Armi, where they are cleaved by Zuc. Some of these phased piRNAs participate in the ping‐pong cycle. Other piRNA precursors, bound by Piwi and cleaved by Zuc, generate Piwi‐piRNAs that are subsequently transported into the nucleus. (B) piRNA biogenesis in somatic cells. piRNA precursor transcripts derived from uni‐strand piRNA clusters, such as flamenco, undergo canonical splicing, 5′ capping and polyadenylation. These processed transcripts are then exported through a complex involving the exon junction complex and Nxf1–Nxt1 complex, which interacts with nucleoporins Nup54 and Nup58 to reach the Yb bodies. Yb binds to these piRNA precursor transcripts and recruits Armi to the Yb bodies, where Piwi binds to the 5′ end of piRNA precursor transcripts after cleavage by a nuclease. Once piRNA precursor transcripts are translocated to the mitochondrial outer membrane, Piwi and Zucchini cleave them to produce Piwi‐bound phased piRNAs. These mature piRNAs are imported into the nucleus in complex with Piwi. Figures in A and B are adapted from Ref. [2] with some modification, with permission. (C) Conservation of processing machinery and the related proteins among Drosophila species. Both ping‐pong‐mediated piRNA biogenesis in germline cells and Yb‐dependent piRNA biogenesis in somatic cells are generally conserved among Drosophila species. However, evolutionary changes have led to the loss of components critical for piRNA biogenesis. For instance, Yb has been independently lost in Drosophila eugracilis and the obscura group. In addition, D. eugracilis has lost Ago3, adopting a ping‐pong‐independent mechanism for piRNA production, whereas the obscura group retains Ago3 and the ping‐pong cycle. Figure in C is adapted from Ref. [100] which is copyrighted under a CC‐BY‐4.0 license, with some modification.

Fig. 3

Conservation of LOTUS and Tudor domains of the TDRD5 and TDRD7. (A) Schematic representations of TDRD 5 and TDRD7 in Drosophila melanogaster. (B) Homology alignment of LOTUS domain of TDRD5 and TDRD7 across metazoans. Accession numbers for TDRD5: Hydra vulgaris: XM047278614.1, Parasteatoda tepidariorum: XM016054396, XM043043873, D. melanogaster: FBgn0033921 [A1Z9P1], Bombyx mori: A0A8R2M345, Danio rerio: A0A8M9PDW1, Xenopus laevis: NP001090599.1 [A1L1H3], Callithrix jacchus: ENSCJAG00000008098 [F7IN5], Homo sapiens: XP054190703.1, Mus musculus: NP001264659.1, Rattus norvegicus: A0A0H2UHC6. TDRD7: Strongylocentrotus purpuratus: XP011669388, H. vulgaris: XM047289989.1, Apis mellifera: XM006562, Plutella xylostella: XP037969050, B. mori: A0A8R2C856, Aedes aegypti: XP0016544, PD D. melanogaster: FBpp0290425, PB D. melanogaster: FBpp0085592, Da. rerio: NP998270.1 [A6NAF9], X. laevis: NP001084569.2 [Q6NU04], C. jacchus: XP002743155.1 [F7HUL2], H. sapiens: NP001289813.1 [Q8NHU6], M. musculus: NP001277404.1 [Q8K1H1], R. norvegicus: NP620226 [Q9R1R4]. (C–E) Homology alignment and a phylogenetic tree of the Tudor domains of TDRD5 and TDRD7 across metazoans generated by clustalw along with itol. Note that TDRD5 homologs typically contain only one Tudor domain, whereas TDRD7 homologs generally harbor three Tudor domains. This structural variance accounts for the observed gaps and differences in domain conservation among the species analyzed. (F) Names of various species used for homology alignment in TDRD5 and TDRD7.

Fig. 4

Function of proteins containing IDR in the piRNA pathways. (A) FRAP analysis demonstrating that IDR of Tej facilitates Vas mobility in Drosophila ovaries. The fluorescence intensity of Vas‐mCherry, co‐expressed with either full‐length Tej (Tej‐FL) (green) or Tej lacking IDR (Tej‐ΔIDR) (magenta), is plotted over time after bleaching. (B) Sequential images of Drosophila nuage show the recovery of the Vas‐mCherry fluorescence intensity in the presence of Tej‐FL or Tej‐ΔIDR before and after photobleaching (marked by dotted white circles). Scale bar indicates 5 μm. (C, E, G) Schematic diagrams illustrate the domain structures of Tej, Yb, and Panx proteins (upper, each panel). IUPRED profiles for these proteins are displayed below (lower, each panel), with the y‐axis representing IUPRED scores. Scores above 0.5 are highlighted in orange. The x‐axis represents the amino acid positions. (D) A schematic model illustrating the function of Tej in ping‐pong piRNA processing. Tej recruits Spn‐E to the cytoplasmic perinuclear nuage via its eSRS motif. In addition, Tej recruits Vas through the LOTUS domain and modulates Vas dynamics through its IDR. (F) A schematic model illustrating the interactions between Yb, Armi, SoYb and Vret within the Yb bodies. Yb forms a homodimer and exhibits sensitivity to 1,6‐hexanediol. (H) A schematic model of the SFiNX complex involved in TE silencing, composed of Panx, Ctp, Nxf2, and Nxt1. This complex forms a DNA‐ or RNA‐dependent granule structure. Figures in A–D are adapted from Ref. [111], which is copyrighted under a CC‐BY‐4.0 license.