Emerging roles and functional mechanisms of PIWI-interacting RNAs.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with proteins of the PIWI clade of the Argonaute family. First identified in animal germ line cells, piRNAs have essential roles in germ line development. The first function of PIWI-piRNA complexes to be described was the silencing of transposable elements, which is crucial for maintaining the integrity of the germ line genome. Later studies provided new insights into the functions of PIWI-piRNA complexes by demonstrating that they regulate protein-coding genes. Recent studies of piRNA biology, including in new model organisms such as golden hamsters, have deepened our understanding of both piRNA biogenesis and piRNA function. In this Review, we discuss the most recent advances in our understanding of piRNA biogenesis, the molecular mechanisms of piRNA function and the emerging roles of piRNAs in germ line development mainly in flies and mice, and in infertility, cancer and neurological diseases in humans.

Activating translation with phase separation.

Ribonucleoprotein granules allow activation of translation to complete mouse spermatogenesis.

Functions of PIWI Proteins in Gene Regulation: New Arrows Added to the piRNA Quiver.

Piwi-interacting RNAs (piRNAs) and PIWI proteins play key functions in a wide range of biological and developmental processes through the regulation of cellular mRNAs, in addition to their role in transposable element (TE) repression. Evolutionary studies indicate that these PIWI functions in mRNA regulatory programs, occurring in both germ and somatic cells, are ancestral. Recent advances have widely expanded our understanding of these functions of PIWI proteins, identifying new mechanisms of action and strengthening their importance through their conservation in distant species. In this review, we discuss the latest findings regarding piRNA/PIWI-dependent mRNA decay in germ cells and during the maternal-to-zygotic transition in embryos combined with new modes of action of PIWI proteins in mRNA stabilization and translational activation and piRNA-independent roles of PIWI proteins in cancer.

The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm.

Piwi-interacting RNAs (piRNAs) and PIWI proteins are essential in germ cells to repress transposons and regulate mRNAs. In Drosophila, piRNAs bound to the PIWI protein Aubergine (Aub) are transferred maternally to the embryo and regulate maternal mRNA stability through two opposite roles. They target mRNAs by incomplete base pairing, leading to their destabilization in the soma and stabilization in the germ plasm. Here, we report a function of Aub in translation. Aub is required for translational activation of nanos mRNA, a key determinant of the germ plasm. Aub physically interacts with the poly(A)-binding protein (PABP) and the translation initiation factor eIF3. Polysome gradient profiling reveals the role of Aub at the initiation step of translation. In the germ plasm, PABP and eIF3d assemble in foci that surround Aub-containing germ granules, and Aub acts with eIF3d to promote nanos translation. These results identify translational activation as a new mode of mRNA regulation by Aub, highlighting the versatility of PIWI proteins in mRNA regulation.

Shaping of Drosophila Neural Cell Lineages Through Coordination of Cell Proliferation and Cell Fate by the BTB-ZF Transcription Factor Tramtrack-69.

Cell diversity in multicellular organisms relies on coordination between cell proliferation and the acquisition of cell identity. The equilibrium between these two processes is essential to assure the correct number of determined cells at a given time at a given place. Using genetic approaches and correlative microscopy, we show that Tramtrack-69 (Ttk69, a Broad-complex, Tramtrack and Bric-à-brac - Zinc Finger (BTB-ZF) transcription factor ortholog of the human promyelocytic leukemia zinc finger factor) plays an essential role in controlling this balance. In the Drosophila bristle cell lineage, which produces the external sensory organs composed by a neuron and accessory cells, we show that ttk69 loss-of-function leads to supplementary neural-type cells at the expense of accessory cells. Our data indicate that Ttk69 (1) promotes cell cycle exit of newborn terminal cells by downregulating CycE, the principal cyclin involved in S-phase entry, and (2) regulates cell-fate acquisition and terminal differentiation, by downregulating the expression of hamlet and upregulating that of Suppressor of Hairless, two transcription factors involved in neural-fate acquisition and accessory cell differentiation, respectively. Thus, Ttk69 plays a central role in shaping neural cell lineages by integrating molecular mechanisms that regulate progenitor cell cycle exit and cell-fate commitment.

aPKC-mediated displacement and actomyosin-mediated retention polarize Miranda in Drosophila neuroblasts.

Cell fate assignment in the nervous system of vertebrates and invertebrates often hinges on the unequal distribution of molecules during progenitor cell division. We address asymmetric fate determinant localization in the developing Drosophila nervous system, specifically the control of the polarized distribution of the cell fate adapter protein Miranda. We reveal a step-wise polarization of Miranda in larval neuroblasts and find that Miranda's dynamics and cortical association are differently regulated between interphase and mitosis. In interphase, Miranda binds to the plasma membrane. Then, before nuclear envelope breakdown, Miranda is phosphorylated by aPKC and displaced into the cytoplasm. This clearance is necessary for the subsequent establishment of asymmetric Miranda localization. After nuclear envelope breakdown, actomyosin activity is required to maintain Miranda asymmetry. Therefore, phosphorylation by aPKC and differential binding to the actomyosin network are required at distinct phases of the cell cycle to polarize fate determinant localization in neuroblasts.

Maintenance of Miranda Localization in Drosophila Neuroblasts Involves Interaction with the Cognate mRNA.

How cells position their proteins is a key problem in cell biology. Targeting mRNAs to distinct regions of the cytoplasm contributes to protein localization by providing local control over translation. Here, we reveal that an interdependence of a protein and cognate mRNA maintains asymmetric protein distribution in mitotic Drosophila neural stem cells. We tagged endogenous mRNA or protein products of the gene miranda that is required for fate determination with GFP. We find that the mRNA localizes like the protein it encodes in a basal crescent in mitosis. We then used GFP-specific nanobodies fused to localization domains to alter the subcellular distribution of the GFP-tagged mRNA or protein. Altering the localization of the mRNA resulted in mislocalization of the protein and vice versa. Protein localization defects caused by mislocalization of the cognate mRNA were rescued by introducing untagged mRNA coding for mutant non-localizable protein. Therefore, by combining the MS2 system and subcellular nanobody expression, we uncovered that maintenance of Mira asymmetric localization requires interaction with the cognate mRNA.

Escargot and Scratch regulate neural commitment by antagonizing Notch activity in Drosophila sensory organs.

During Notch (N)-mediated binary cell fate decisions, cells adopt two different fates according to the levels of N pathway activation: an Noff-dependent or an Non-dependent fate. How cells maintain these N activity levels over time remains largely unknown. We address this question in the cell lineage that gives rise to the Drosophila mechanosensory organs. In this lineage a primary precursor cell undergoes a stereotyped sequence of oriented asymmetric cell divisions and transits through two neural precursor states before acquiring a neuron identity. Using a combination of genetic and cell biology strategies, we show that Escargot and Scratch, two transcription factors belonging to the Snail superfamily, maintain Noff neural commitment by directly blocking the transcription of N target genes. We propose that Snail factors act by displacing proneural transcription activators from DNA binding sites. As such, Snail factors maintain the Noff state in neural precursor cells by buffering any ectopic variation in the level of N activity. Since Escargot and Scratch orthologs are present in other precursor cells, our findings are fundamental for understanding precursor cell fate acquisition in other systems.