Daedalus and Gasz recruit Armitage to mitochondria, bringing piRNA precursors to the biogenesis machinery

ABSTRACTThe piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialised machinery that converts long single-stranded precursors into small RNAs of ~25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb-body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and for the correct localisation of the indispensable piRNA biogenesis factor, Armitage (Armi). We find that Gasz and Daed interact with each other and likely provide a mitochondrial “anchoring platform” to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb-bodies in a manner that depends upon Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing, namely how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.

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Intergenerational metabolic priming by sperm piRNAs

10.1101/2021.03.29.436592 ◽

2021 ◽

Author(s):

Adelheid Lempradl ◽

Unn Kugelberg ◽

Mary Iconomou ◽

Ian Beddows ◽

Daniel Nätt ◽

...

Keyword(s):

Small Rna ◽

Small Rnas ◽

Target Gene ◽

Epigenetic Inheritance ◽

Mature Sperm ◽

Transcriptional Reprogramming ◽

Complementary Sequences ◽

Specific Manner ◽

Dietary Sugar ◽

Pirna Pathway

Preconception parental environment can reproducibly program offspring phenotype without altering the DNA sequence, yet the mechanisms underpinning this epigenetic inheritance remains elusive. Here, we demonstrate the existence of an intact piRNA-pathway in mature Drosophila sperm and show that pathway modulation alters offspring gene transcription in a sequence-specific manner. We map a dynamic small RNA content in developing sperm and find that the mature sperm carry a highly distinct small RNA cargo. By biochemical pulldown, we identify a small RNA subset bound directly to piwi protein. And, we show that piRNA-pathway controlled sperm small RNAs are linked to target gene repression in offspring. Critically, we find that full piRNA-pathway dosage is necessary for the intergenerational metabolic and transcriptional reprogramming events triggered by high paternal dietary sugar. These data provide a direct link between regulation of endogenous mature sperm small RNAs and transcriptional programming of complementary sequences in offspring. Thus, we identify a novel mediator of paternal intergenerational epigenetic inheritance.

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The Caenorhabditis elegans TDRD5/7-like protein, LOTR-1, interacts with the helicase ZNFX-1 to balance epigenetic signals in the germline

10.1101/2021.06.18.448978 ◽

2021 ◽

Author(s):

Elisabeth A Marnik ◽

Miguel Vasconcelos Almeida ◽

P Giselle Cipriani ◽

George Chung ◽

Edoardo Caspani ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Small Rna ◽

Small Rnas ◽

Brood Size ◽

Homologous Proteins ◽

P Granules ◽

Transposon Silencing ◽

Tudor Domain ◽

Germ Granules

LOTUS and Tudor domain containing proteins have critical roles in the germline. Proteins that contain these domains, such as Tejas/Tapas in Drosophila, help localize Vasa to the germ granules and facilitate piRNA-mediated transposon silencing. The homologous proteins in mammals, TDRD5 and TDRD7, are required during spermiogenesis. Until now, proteins containing both LOTUS and Tudor domains in Caenorhabditis elegans have remained elusive. Here we describe LOTR-1 (D1081.7), which derives its name from its LOTUS and Tudor domains. Interestingly, LOTR-1 docks next to P granules to colocalize with the broadly conserved Z-granule helicase, ZNFX-1. LOTR-1's Z-granule association requires its Tudor domain, but both LOTUS and Tudor deletions affect brood size when coupled with a knockdown of the Vasa homolog glh-1. In addition to interacting with the germ-granule components WAGO-1, PRG-1 and DEPS-1, we identified a Tudor-dependent association with ZNFX-1. Like znfx-1 mutants, lotr-1 mutants lose small RNAs from the 3' ends of WAGO and Mutator targets, reminiscent of the loss of piRNAs from the 3' ends of piRNA precursor transcripts in mouse Tdrd5 mutants. Our work suggests that LOTR-1 acts in a conserved mechanism that brings small RNA generating mechanisms towards the 3' ends of small RNA templates or precursors.

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Specialization of the Drosophila nuclear export family protein, Nxf3, for piRNA precursor export

10.1101/716258 ◽

2019 ◽

Cited By ~ 1

Author(s):

Emma Kneuss ◽

Marzia Munafò ◽

Evelyn L. Eastwood ◽

Undine-Sophie Deumer ◽

Jonathan B. Preall ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Small Rna ◽

Nuclear Export ◽

Mrna Export ◽

Transposon Silencing ◽

Family Protein ◽

Pirna Pathway ◽

Harmful Effects ◽

Shed Light ◽

Mature Mrnas

AbstractThe piRNA pathway is a conserved, small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilisation. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use non-canonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by Nuclear export factor 1. Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila Nuclear export factor family protein, Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We find that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.

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Nematode Small RNA Pathways in the Absence of piRNAs

10.1101/2021.07.23.453445 ◽

2021 ◽

Author(s):

Maxim Zagoskin ◽

Jianbin Wang ◽

Ashley T. Neff ◽

Giovana M.B. Veronezi ◽

Richard E. Davis

Keyword(s):

Small Rna ◽

Small Rnas ◽

Parasitic Nematode ◽

Repetitive Sequences ◽

C Elegans ◽

Early Embryos ◽

Specific Mrnas ◽

Rna Pathways ◽

Pirna Pathway ◽

Mature Mrnas

Small RNA pathways play diverse regulatory roles in the nematode C. elegans. However, our understanding of small RNA pathways, their conservation, and their roles in other nematodes is limited. Here, we analyzed small RNA pathways in the parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that are associated with secondary 5'-triphosphate small RNAs (22-24G-RNAs). These Ascaris WAGOs and their small RNAs target repetitive sequences (WAGO-1, WAGO-2, WAGO-3, and NRDE-3) or mature mRNAs (CSR-1, NRDE-3, and WAGO-3) and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Ascaris CSR-1 likely functions to "license" gene expression in the absence of an Ascaris piRNA pathway. Ascaris ALG-4 and its associated 26G-RNAs target and appear to repress specific mRNAs during meiosis in the testes. Notably, Ascaris WAGOs (WAGO-3 and NRDE-3) small RNAs change their targets between repetitive sequences and mRNAs during spermatogenesis or in early embryos illustrating target plasticity of these WAGOs. We provide a unique and comprehensive view of mRNA and small RNA expression throughout nematode spermatogenesis that illustrates the dynamics and flexibility of small RNA pathways. Overall, our study provides key insights into the conservation and divergence of nematode small RNA pathways.

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Glucose metabolism in cancer cells: immunogold localization of hexokinase to the outer mitochondrial membrane

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141597 ◽

1995 ◽

Vol 53 ◽

pp. 1044-1045

Author(s):

Krishan K. Arora ◽

Glenn L. Decker ◽

Peter L. Pedersen

Keyword(s):

Tumor Cells ◽

Mitochondrial Membrane ◽

Present Report ◽

Large Fraction ◽

Mitochondrial Fraction ◽

Immunogold Labeling ◽

Outer Mitochondrial Membrane ◽

Immunogold Localization ◽

Hexokinase Activity ◽

Normal Tissues

Hexokinase (ATP: D-hexose 6-phophotransferase EC 2.7.1.1) is the first enzyme of the glycolytic pathway which commits glucose to catabolism by catalyzing the phosphorylation of glucose with ATP. Previous studies have shown diat hexokinase activity is markedly elevated in rapidly growing tumor cells exhibiting high glucose catabolic rates. A large fraction (50-80%) of this enzyme activity is bound to the mitochondrial fraction (1,2) where it has preferred access to ATP (3). In contrast,the hexokinase activity of normal tissues is quite low, with one exception being brain which is a glucose-utilizing tissue (4). Biochemical evidence involving rigorous subfractionation studies have revealed striking differences between the subcellular distribution of hexokinase in normal and tumor cells [See review by Arora et al (4)].In the present report, we have utilized immunogold labeling techniques to evaluate die subcellular localization of hexokinase in highly glycolytic AS-30D hepatoma cells and in the tissue of its origin, i.e., rat liver.

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