scholarly journals Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila

2021 ◽  
Author(s):  
Evelyn L Eastwood ◽  
Kayla A Jara ◽  
Susanne Bornelöv ◽  
Marzia Munafò ◽  
Vasileios Frantzis ◽  
...  
Keyword(s):  
Transcriptional Silencing ◽  
Transcriptional Gene Silencing ◽  
Dynein Light Chain ◽  
Heterochromatin Formation ◽  
Transposon Silencing ◽  
Cellular Machinery ◽  
Transposon Insertions ◽  
Pirna Pathway ◽  
Fundamental Requirement ◽  
Rna And Dna

In animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. In Drosophila ovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through the Panoramix-induced co-transcriptional silencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. Here, we report that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. We show that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex.

scholarly journals Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Evelyn L Eastwood ◽  
Kayla A Jara ◽  
Susanne Bornelöv ◽  
Marzia Munafò ◽  
Vasileios Frantzis ◽  
...  
Keyword(s):  
Genome Integrity ◽  
Transcriptional Gene Silencing ◽  
Dynein Light Chain ◽  
Heterochromatin Formation ◽  
Transposon Silencing ◽  
Cellular Machinery ◽  
Transposon Insertions ◽  
Pirna Pathway ◽  
Fundamental Requirement ◽  
Rna And Dna

In animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. In Drosophila ovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through the Panoramix-induced co-transcriptional silencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. Here, we report that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. We show that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex.

scholarly journals Xrn1p acts at multiple steps in the budding-yeast RNAi pathway to enhance the efficiency of silencing

2020 ◽  
Author(s):  
Matthew A Getz ◽  
David E Weinberg ◽  
Ines A Drinnenberg ◽  
Gerald R Fink ◽  
David P Bartel
Keyword(s):  
Gene Silencing ◽  
Budding Yeast ◽  
Genetic Selection ◽  
Rnai Pathway ◽  
Transcriptional Gene Silencing ◽  
Heterochromatin Formation ◽  
Post Transcriptional Gene Silencing ◽  
Passenger Strand ◽  
Transposon Silencing ◽  
Target Rna

Abstract RNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae, RNAi is present in other budding-yeast species, including Naumovozyma castellii, which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection. This selection identified Xrn1p, the cytoplasmic 5′-to-3′ exoribonuclease, as a cofactor of RNAi in budding yeast. Deletion of XRN1 impaired gene silencing in N. castellii, and this impaired silencing was attributable to multiple functions of Xrn1p, including affecting the composition of siRNA species in the cell, influencing the efficiency of siRNA loading into Argonaute, degradation of cleaved passenger strand and degradation of sliced target RNA.

scholarly journals The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation

2019 ◽  
Author(s):  
Julia Batki ◽  
Jakob Schnabl ◽  
Juncheng Wang ◽  
Dominik Handler ◽  
Veselin I. Andreev ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Transcriptional Silencing ◽  
Genome Integrity ◽  
Rna Transport ◽  
Heterochromatin Formation ◽  
Genome Defense ◽  
Molecular Events ◽  
Pirna Pathway ◽  
Transport Receptor

ABSTRACTThe PIWI-interacting RNA (piRNA) pathway protects animal genome integrity in part through establishing repressive heterochromatin at transposon loci. Silencing requires piRNA-guided targeting of nuclear PIWI proteins to nascent transposon transcripts, yet the subsequent molecular events are not understood. Here, we identify SFiNX (Silencing Factor interacting Nuclear eXport variant), an interdependent protein complex required for Piwi-mediated co-transcriptional silencing in Drosophila. SFiNX consists of Nxf2-Nxt1, a gonad-specific variant of the heterodimeric mRNA export receptor Nxf1-Nxt1, and the Piwi-associated protein Panoramix. SFiNX mutant flies are sterile and exhibit transposon de-repression because piRNA-loaded Piwi is unable to establish heterochromatin. Within SFiNX, Panoramix recruits the heterochromatin effectors, while the RNA binding Nxf2 protein licenses co-transcriptional silencing. Our data reveal how Nxf2 evolved from an RNA transport receptor into a co-transcriptional silencing factor. Thus, NXF-variants, which are abundant in metazoans, can have diverse molecular functions and might have been co-opted for host genome defense more broadly.

scholarly journals Xrn1p Acts at Multiple Steps in the Budding-Yeast RNAi Pathway to Enhance the Efficiency of Silencing

2019 ◽  
Author(s):  
Matthew A. Getz ◽  
David E. Weinberg ◽  
Ines A. Drinnenberg ◽  
Gerald R. Fink ◽  
David P. Bartel
Keyword(s):  
Gene Silencing ◽  
Budding Yeast ◽  
Genetic Selection ◽  
Rnai Pathway ◽  
Transcriptional Gene Silencing ◽  
Heterochromatin Formation ◽  
Post Transcriptional Gene Silencing ◽  
Passenger Strand ◽  
Transposon Silencing ◽  
Target Rna

AbstractRNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation, and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae, RNAi is present in other budding-yeast species, including Naumovozyma castellii, which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection. This selection identified Xrn1p, the cytoplasmic 5′-to-3′ exoribonuclease, as a cofactor of RNAi in budding yeast. Deletion of XRN1 impaired gene silencing in N. castellii, and this impaired silencing was attributable to multiple functions of Xrn1p, including affecting the composition of siRNA species in the cell, influencing the efficiency of siRNA loading into Argonaute, degradation of cleaved passenger strand, and degradation of sliced target RNA.

scholarly journals Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing

2019 ◽  
Author(s):  
Kensaku Murano ◽  
Yuka W. Iwasaki ◽  
Hirotsugu Ishizu ◽  
Akane Mashiko ◽  
Aoi Shibuya ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Transcriptional Silencing ◽  
List Type ◽  
Dependent Manner ◽  
Heterochromatin Formation ◽  
Protein Levels ◽  
Nuclear Rna ◽  
Rna Export ◽  
Nuclear Rna Export ◽  
Pirna Pathway

SummaryThe PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-p15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Nxf2-Panx-p15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway.HighlightsNxf2 plays an essential role in the Piwi–piRNA pathwayFormation of Piwi-Panx-Nxf2-p15 (PPNP) complexes stabilizes both Panx and Nxf2The PPNP complex triggers transcriptional silencing before heterochromatin formationNxf2 directly binds to target transcripts in a Piwi-dependent manner

scholarly journals A Pandas complex adapted for piRNA-guided transposon silencing

2019 ◽  
Author(s):  
Kang Zhao ◽  
Sha Cheng ◽  
Na Miao ◽  
Ping Xu ◽  
Xiaohua Lu ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Pore ◽  
Nuclear Retention ◽  
Heterochromatin Formation ◽  
Transposon Silencing ◽  
Uba Domain ◽  
Rna Export ◽  
Nascent Transcripts ◽  
Functional Analyses ◽  
Pirna Pathway

AbstractThe repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila ovaries, Panoramix (Panx) enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. Here, we show that Panx functions together with a germline specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), as a ternary complex to suppress transposon expression. Structural and functional analyses demonstrate that dNxf2 binds Panx via its UBA domain, which plays an important role in transposon silencing. Unexpectedly, dNxf2 interacts directly with dNxf1 (TAP), a general nuclear export factor. As a result, dNxf2 prevents dNxf1 from binding to the FG repeats of the nuclear pore complex, a process required for proper RNA export. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. Therefore, we propose that dNxf2 may function as a Pandas (Panoramix-dNxf2 dependent TAP/p15 silencing) complex, which counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. Our findings may have broader implications for understanding how RNA metabolism modulates epigenetic gene silencing and heterochromatin formation.

scholarly journals The molecular principles of Piwi-mediated co-transcriptional silencing through the dimeric SFiNX complex

2021 ◽  
Author(s):  
Jakob Schnabl ◽  
Juncheng Wang ◽  
Ulrich Hohmann ◽  
Maja Gehre ◽  
Julia Batki ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transcriptional Silencing ◽  
General Mechanism ◽  
Nucleic Acid Binding ◽  
Dynein Light Chain ◽  
Dimerization Domain ◽  
Heterochromatin Formation ◽  
Condensate Formation

Nuclear Argonaute proteins, guided to nascent target RNAs by their bound small RNAs, elicit co-transcriptional silencing through heterochromatin formation at transposon insertions and repetitive genomic loci. The molecular mechanisms involved in this process are incompletely understood. Here, we propose that the SFiNX complex, a silencing mediator downstream of nuclear Piwi-piRNA complexes in Drosophila, enables co-transcriptional silencing via the formation of molecular condensates. Condensate formation is stimulated by nucleic acid binding and requires SFiNX dimerization, mediated by the dynein light chain protein, LC8/Cutup. LC8's function within SFiNX can be bypassed with a heterologous dimerization domain, suggesting that dimerization is a constitutive feature of SFiNX. Mutations preventing LC8- mediated SFiNX dimerization result in loss of condensate formation in vitro and inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. Formation of molecular condensates might be a general mechanism that underlies effective heterochromatin establishment at small RNA target loci in a co-transcriptional manner.

scholarly journals Post-transcriptional gene silencing triggers dispensable DNA methylation in gene body in Arabidopsis

2019 ◽  
Vol 47 (17) ◽  
pp. 9104-9114 ◽  
Author(s):  
Christelle Taochy ◽  
Agnès Yu ◽  
Nicolas Bouché ◽  
Nathalie Bouteiller ◽  
Taline Elmayan ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Gene Silencing ◽  
De Novo ◽  
Transcriptional Silencing ◽  
Transcriptional Gene Silencing ◽  
Gene Body ◽  
Coding Sequence ◽  
Post Transcriptional Gene Silencing ◽  
Methylation Signal ◽  
Rule Out

Abstract Spontaneous post-transcriptional silencing of sense transgenes (S-PTGS) is established in each generation and is accompanied by DNA methylation, but the pathway of PTGS-dependent DNA methylation is unknown and so is its role. Here we show that CHH and CHG methylation coincides spatially and temporally with RDR6-dependent products derived from the central and 3′ regions of the coding sequence, and requires the components of the RNA-directed DNA methylation (RdDM) pathway NRPE1, DRD1 and DRM2, but not CLSY1, NRPD1, RDR2 or DCL3, suggesting that RDR6-dependent products, namely long dsRNAs and/or siRNAs, trigger PTGS-dependent DNA methylation. Nevertheless, none of these RdDM components are required to establish S-PTGS or produce a systemic silencing signal. Moreover, preventing de novo DNA methylation in non-silenced transgenic tissues grafted onto homologous silenced tissues does not inhibit the triggering of PTGS. Overall, these data indicate that gene body DNA methylation is a consequence, not a cause, of PTGS, and rule out the hypothesis that a PTGS-associated DNA methylation signal is transmitted independent of a PTGS signal.

scholarly journals Assembly and Function of Gonad-Specific Non-Membranous Organelles in Drosophila piRNA Biogenesis

2019 ◽  
Vol 5 (4) ◽  
pp. 52 ◽  
Author(s):  
Shigeki Hirakata ◽  
Mikiko C. Siomi
Keyword(s):  
Dna Damage ◽  
Gonadal Development ◽  
Mitochondrial Membranes ◽  
Animal Life ◽  
Transposon Silencing ◽  
Dna Elements ◽  
Non Coding Rnas ◽  
And Function ◽  
Pirna Pathway

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that repress transposons in animal germlines. This protects the genome from the invasive DNA elements. piRNA pathway failures lead to DNA damage, gonadal development defects, and infertility. Thus, the piRNA pathway is indispensable for the continuation of animal life. piRNA-mediated transposon silencing occurs in both the nucleus and cytoplasm while piRNA biogenesis is a solely cytoplasmic event. piRNA production requires a number of proteins, the majority of which localize to non-membranous organelles that specifically appear in the gonads. Other piRNA factors are localized on outer mitochondrial membranes. In situ RNA hybridization experiments show that piRNA precursors are compartmentalized into other non-membranous organelles. In this review, we summarize recent findings about the function of these organelles in the Drosophila piRNA pathway by focusing on their assembly and function.

Lysine-specific histone demethylase LSD1 and the dynamic control of chromatin

2013 ◽  
Vol 394 (8) ◽  
pp. 1019-1028 ◽  
Author(s):  
Thomas Rudolph ◽  
Stefanie Beuch ◽  
Gunter Reuter
Keyword(s):  
Molecular Analysis ◽  
Transcriptional Repression ◽  
Amine Oxidase ◽  
Protein Complexes ◽  
Dynamic Control ◽  
Histone Demethylase ◽  
Model Organisms ◽  
Transcriptional Gene Silencing ◽  
Heterochromatin Formation ◽  
Enzyme Complexes

Abstract The flavin adenine dinucleotide-dependent amine oxidase LSD1 is the first molecularly defined histone demethylase, which specifically demethylates H3K4me1/me2. The enzyme dynamically controls a large variety of biological processes and is associated with protein complexes controlling transcriptional repression and activation. Molecular analysis of the Drosophila LSD1 homolog revealed new insights into the epigenetic control of heterochromatin formation during early embryogenesis, the establishment of transcriptional gene silencing and the epigenetic mechanisms associated with the maintenance of stem cell identity in primordial germline cells. This review summarizes our recent knowledge about the control of enzymatic activity and molecular function of LSD1 enzyme complexes in different model organisms including Schizosaccharomyces pombe, Drosophila and mammals. Finally, new developments in applied cancer research based on molecular analysis of LSD1 in cancer cells are discussed.

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