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

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).

HemK2 functions for sufficient protein synthesis and RNA stability through eRF1 methylation during Drosophila oogenesis.

HemK2 is a highly conserved methyltransferase, but the identification of its genuine substrates has been controversial, and its biological importance in higher organisms remains unclear. We elucidate the role of HemK2 in the methylation of eukaryotic Release Factor 1 (eRF1), a process that is essential for female germline development in Drosophila melanogaster. Knockdown of hemK2 in the germline cells (hemK2-GLKD) induces apoptosis, accompanied by a pronounced decrease in both eRF1 methylation and protein synthesis.

Microbes control Drosophila germline stem cell increase and egg maturation through hormonal pathways.

Reproduction is highly dependent on environmental and physiological factors including nutrition, mating stimuli and microbes. Among these factors, microbes facilitate vital functions for host animals such as nutritional intake, metabolic regulation, and enhancing fertility under poor nutrition conditions. However, detailed molecular mechanisms by which microbes control germline maturation, leading to reproduction, remain largely unknown.

Tejas functions as a core component in nuage assembly and precursor processing in Drosophila piRNA biogenesis.

PIWI-interacting RNAs (piRNAs), which protect genome from the attack by transposons, are produced and amplified in membraneless granules called nuage. In Drosophila, PIWI family proteins, Tudor-domain-containing (Tdrd) proteins, and RNA helicases are assembled and form nuage to ensure piRNA production. However, the molecular functions of the Tdrd protein Tejas (Tej) in piRNA biogenesis remain unknown.

miRNA/siRNA-directed pathway to produce noncoding piRNAs from endogenous protein-coding regions ensures spermatogenesis.

PIWI-interacting RNA (piRNA) pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the underlying piRNA biogenesis mechanisms remain elusive. Here, we show that endogenous protein coding sequences (CDSs), which are normally used for translation, serve as origins of noncoding piRNA biogenesis in testes.

Nondomain biopolymers: Flexible molecular strategies to acquire biological functions.

A long-standing assumption in molecular biology posits that the conservation of protein and nucleic acid sequences emphasizes the functional significance of biomolecules. These conserved sequences fold into distinct secondary and tertiary structures, enable highly specific molecular interactions, and regulate complex yet organized molecular processes within living cells. However, recent evidence suggests that biomolecules can also function through primary sequence regions that lack conservation across species or gene families.

The Tudor Domain-Containing Protein, Kotsubu (CG9925), Localizes to the Nuage and Functions in piRNA Biogenesis in .

Silencing of transposable elements (TEs) by Piwi-interacting RNAs (piRNAs) is crucial for maintaining germline genome integrity and fertility in animals. To repress TEs, PIWI clade Argonaute proteins cooperate with several Tudor domain-containing (Tdrd) proteins at membraneless perinuclear organelles, called nuage, to produce piRNAs to repress transposons. Here, we identify and characterize Kotsubu (Kots), one of the Tudor domain-containing protein-1 (Tdrd1) orthologs, encoded by the gene, that localizes to the nuage in gonads.

Modulation of Ago2 Loading by Cyclophilin 40 Endows a Unique Repertoire of Functional miRNAs during Sperm Maturation in Drosophila.

In gene silencing, Hsp90 chaperone machinery assists Argonaute (Ago) binding and unwinding of silencing small RNA (sRNA) duplexes. This enables the formation of effector RNA-induced silencing complex (RISC) that often displays cargo preferences. Hence, in Drosophila, microRNAs (miRNAs) and small-interfering RNAs (siRNAs) are differentially sorted into Ago1-RISC and Ago2-RISC, respectively.

Drosophila MARF1 ensures proper oocyte maturation by regulating nanos expression.

Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. In this study, we examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation.

Heterochromatin protein 1a functions for piRNA biogenesis predominantly from pericentric and telomeric regions in Drosophila.

In metazoan germline, Piwi-interacting RNAs (piRNAs) provide defence against transposons. Piwi-piRNA complex mediates transcriptional silencing of transposons in nucleus. Heterochromatin protein 1a (HP1a) has been proposed to function downstream of Piwi-piRNA complex in Drosophila.

Protecting and Diversifying the Germline.

Gametogenesis represents the most dramatic cellular differentiation pathways in both female and male flies. At the genome level, meiosis ensures that diploid germ cells become haploid gametes. At the epigenome level, extensive changes are required to turn on and shut off gene expression in a precise spatiotemporally controlled manner.

The piRNA pathway is developmentally regulated during spermatogenesis in Drosophila.

PIWI-interacting RNAs (piRNAs) are predominantly produced in animal gonads to suppress transposons during germline development. Our understanding about the piRNA biogenesis and function is predominantly from studies of the Drosophila female germline. piRNA pathway function in the male germline, however, remains poorly understood.

A piece of the pi(e): The diverse roles of animal piRNAs and their PIWI partners.

Small non-coding RNAs are indispensable to many biological processes. A class of endogenous small RNAs, termed PIWI-interacting RNAs (piRNAs) because of their association with PIWI proteins, has known roles in safeguarding the genome against inordinate transposon mobilization, embryonic development, and stem cell regulation, among others. This review discusses the biogenesis of animal piRNAs and their diverse functions together with their PIWI protein partners, both in the germline and in somatic cells, and highlights the evolutionarily conserved aspects of these molecular players in animal biology.

Isolation of Undifferentiated Female Germline Cells from Adult Drosophila Ovaries.

This unit describes a method for isolating undifferentiated, stem cell-like germline cells from adult Drosophila ovaries. Here, we demonstrate that this population of cells can be effectively purified from hand-dissected ovaries in considerably large quantities. Tumor ovaries with expanded populations of undifferentiated germline cells are first removed from fly abdomens and dissociated into a cell suspension with the aid of protease treatment.

The Tudor domain protein Tapas, a homolog of the vertebrate Tdrd7, functions in the piRNA pathway to regulate retrotransposons in germline of Drosophila melanogaster.

Background: Piwi-interacting RNAs (piRNAs) are a special class of small RNAs that provide defense against transposable elements in animal germline cells. In Drosophila, germline piRNAs are thought to be processed at a unique perinuclear structure, the nuage, that houses piRNA pathway proteins including the Piwi clade of Argonaute family proteins, along with several Tudor domain proteins, RNA helicases and nucleases. We previously demonstrated that Tudor domain protein Tejas (Tej), an ortholog of vertebrate Tdrd5, is an important component of the piRNA pathway.

Analysis of Hydra PIWI proteins and piRNAs uncover early evolutionary origins of the piRNA pathway.

To preserve genome integrity, an evolutionarily conserved small RNA-based silencing mechanism involving PIWI proteins and PIWI-interacting RNAs (piRNAs) represses potentially deleterious transposons in animals. Although there has been extensive research into PIWI proteins in bilaterians, these proteins remain to be examined in ancient phyla. Here, we investigated the PIWI proteins Hywi and Hyli in the cnidarian Hydra, and found that both PIWI proteins are enriched in multipotent stem cells, germline stem cells, and in the female germline.

Polo-mediated phosphorylation of Maelstrom regulates oocyte determination during oogenesis in Drosophila.

In Drosophila, Maelstrom is a conserved component of the perinuclear nuage, a germline-unique structure that appears to serve as a site for Piwi-interacting RNA (piRNA) production to repress deleterious transposons. Maelstrom also functions in the nucleus as a transcriptional regulator to repress the expression of microRNA-7, a process that is essential for the proper differentiation of germline stem cells. In this paper, we report another function of Maelstrom in regulating oocyte determination independently of its transposon silencing and germline stem cell differentiation activities.

Isolation of undifferentiated female germline cells from adult Drosophila ovaries.

This unit describes a method for isolating undifferentiated, stem cell-like germline cells from adult Drosophila ovaries. Here, we demonstrate that this population of cells can be effectively purified from hand-dissected ovaries in considerably large quantities. Tumor ovaries with expanded populations of undifferentiated germline cells are first removed from fly abdomens and dissociated into a cell suspension with the aid of protease treatment.

Tudor domain proteins in development.

Tudor domain proteins function as molecular adaptors, binding methylated arginine or lysine residues on their substrates to promote physical interactions and the assembly of macromolecular complexes. Here, we discuss the emerging roles of Tudor domain proteins during development, most notably in the Piwi-interacting RNA pathway, but also in other aspects of RNA metabolism, the DNA damage response and chromatin modification.

piRNA pathway and the potential processing site, the nuage, in the Drosophila germline.

The accurate transfer of genetic material in germline cells during the formation of gametes is important for the continuity of the species. However, animal germline cells face challenges from transposons, which seek to spread themselves in the genome. This review focuses on studies in Drosophila melanogaster on how the genome protects itself from such a mutational burden via a class of gonad-specific small interfering RNAs, known as piRNAs (Piwi-interacting RNAs).

The tudor domain protein kumo is required to assemble the nuage and to generate germline piRNAs in Drosophila.

In Drosophila ovaries, distinct Piwi-interacting RNA (piRNA) pathways defend against transposons in somatic and germline cells. Germline piRNAs predominantly arise from bidirectional clusters and are amplified by the ping-pong cycle. In this study, we characterize a novel Drosophila gene, kumo and show that it encodes a conserved germline piRNA pathway component.

DEAD-box RNA helicase Belle/DDX3 and the RNA interference pathway promote mitotic chromosome segregation.

During mitosis, faithful inheritance of genetic material is achieved by chromosome segregation, as mediated by the condensin I and II complexes. Failed chromosome segregation can result in neoplasm formation, infertility, and birth defects. Recently, the germ-line-specific DEAD-box RNA helicase Vasa was demonstrated to promote mitotic chromosome segregation in Drosophila by facilitating robust chromosomal localization of Barren (Barr), a condensin I component.

Non-coding RNAs enter mitosis: functions, conservation and implications.

Nuage (or commonly known as chromatoid body in mammals) is a conserved germline-specific organelle that has been linked to the Piwi-interacting RNA (piRNA) pathway. piRNAs are a class of gonadal-specific RNAs that are ~23-29 nucleotides in length and protect genome stability by repressing the expression of deleterious retrotransposons. More recent studies in Drosophila have implicated the piRNA pathway in other functions including canalization of embryonic development, regulation of maternal gene expression and telomere protection.