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

Iki, Taichiro; Takami, Moe; Kai, Toshie

doi:10.1016/j.celrep.2020.108380

Cited by 8 publications

(20 citation statements)

References 80 publications

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“…Cyp40 can support CDS-piRNA production both directly through the interaction with Aub (Fig. 3) and indirectly through the modulation of Ago2 loading (25). This study showed that piRNA production can be directed by both siRNAs and miRNAs binding to Ago2 in fly testicular germ line (Fig.…”

Section: Discussionmentioning

confidence: 73%

“…sRNA loading step in RISC formation relies on Hsp70/Hsp90 chaperone machineries (29)(30)(31)(32). In Drosophila, an Hsp90 cochaperone Cyp40 is expressed in testicular germ cells and essential for sperm formation; however, its substrates/clients are elusive besides Ago2 (25). Hence, we performed Cyp40 client screening based on proximity-dependent biotin identification (BioID/TurboID) (33,34), which could be advanced in identifying transient chaperone-client interactions compared to conventional immunoprecipitation analyses (Fig.…”

Section: Aub Is Physically Linked To Cyp40 An Hsp90 Cochaperone Speci...mentioning

confidence: 99%

“…Endo-siRNAs guide Ago2 and direct mRNA cleavage, thereby supporting late spermatogenesis (22)(23)(24). We recently reported a cochaperone associated with Hsp90 machinery, Cyclophilin 40 (Cyp40), is preferentially expressed in male germ line and essential for sperm formation (25). Ago2 binds testis-unique microRNAs (miRNAs) and endo-siRNAs, and Cyp40 increases the miRNA occupancy inside RISCs.…”

Section: Introductionmentioning

confidence: 99%

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

2023

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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 Drosophila melanogaster testes. The product, namely, CDS-piRNAs, formed silencing complexes with Aubergine (Aub) in germ cells. Proximity proteome and functional analyses show that CDS-piRNAs and cluster/TE-piRNAs are distinct species occupying Aub, the former loading selectively relies on chaperone Cyclophilin 40. Moreover, Argonaute 2 (Ago2) and Dicer-2 activities were found critical for CDS-piRNA production. We provide evidence that Ago2-bound short interfering RNAs (siRNAs) and microRNAs (miRNAs) specify precursors to be processed into piRNAs. We further demonstrate that Aub is crucial in spermatid differentiation, regulating chromatins through mRNA cleavage. Collectively, our data illustrate a unique strategy used by male germ line, expanding piRNA repertoire for silencing of endogenous genes during spermatogenesis.

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Section: Discussionmentioning

confidence: 73%

Section: Aub Is Physically Linked To Cyp40 An Hsp90 Cochaperone Speci...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

2023

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“…For the establishment of CG9899/ctr9t KO by CRISPR-Cas9, the left and right homology arms were amplified by pfusion PCR (NEB) using y w genomic DNA as template and primer pairs Ti897/898 and Ti899/900. A fragment containing 3xP3-dsRed marker was amplified using primer pairs Ti673/Mt024 (template: pBlue-cyp40KOarm [67]). These three fragments were cloned into EcoRI-digested pBluescript-II vector using In-Fusion HD cloning kit (Takara).…”

Section: Plasmid Construction Fly Transformation Mutagenesismentioning

confidence: 99%

Paralog-dependent Specialization of Paf1C subunit, Ctr9, for Sex Chromosome Gene Regulation and Male Germline Differentiation in Drosophila

Kai,

Zheng,

Iki

2024

Preprint

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Testis-specific gene regulatory mechanisms govern the differentiation of germ cells into mature sperms. However, the molecular underpinnings are not fully elucidated. Here, we show the subunits forming Polymerase Associated Factor 1 Complex (Paf1C), a transcription regulator conserved across eukaryotes, have their individual paralogs predominantly expressed in Drosophila testes. One of these, namely, Ctr9 paralog enriched in testes (Ctr9t) was found to play a critical and nonredundant role in post-meiotic spermatid differentiation and male fertility in D. melanogaster. A proximity proteome analysis provides evidence that Ctr9t forms complexes with other paralog members. We show endogenous Ctr9t is expressed in germ cells at spermatocyte stages, accumulating in a distinct compartment within the nucleolus. There, Ctr9t co-localizes with Spermatocyte arrest (Sa), a testis-specific paralog of TATA-binding protein (TBP)-associated factor 8 (TAF8). We further demonstrate that ctr9t function is crucial for maintaining Sa in the nucleolus, but not vice versa. Transcriptome profiling reveals that Ctr9t acts as an activator for the set of male fertility genes on Y chromosome, but it also acts as a global repressor of X chromosome genes. Collectively, our results shed light on the nucleolus-associated, paralog-dependent regulation of gene expression from sex chromosomes, which ensures the terminal differentiation of male germ cells.

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“…The plant CYP40 acts as a co-chaperone of Hsp90 to promote microRNA activities by facilitating the binding of small ligands to Ago1 ( 35 ). The CYP40 homolog in Drosophila melanogaster has also been identified as the Hsp90 co-chaperone, which is essential for spermatogenesis and modulation of Ago2-RISC formation ( 36 ). Nevertheless, other possible chaperone functions of CYPs remain to be explored.…”

Section: Introductionmentioning

confidence: 99%

Cyclophilin acts as a ribosome biogenesis factor by chaperoning the ribosomal protein (PlRPS15) in filamentous fungi

Xie

Wang

et al. 2021

Nucleic Acids Research

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The rapid transport of ribosomal proteins (RPs) into the nucleus and their efficient assembly into pre-ribosomal particles are prerequisites for ribosome biogenesis. Proteins that act as dedicated chaperones for RPs to maintain their stability and facilitate their assembly have not been identified in filamentous fungi. PlCYP5 is a nuclear cyclophilin in the nematophagous fungus Purpureocillium lilacinum, whose expression is up-regulated during abiotic stress and nematode egg-parasitism. Here, we found that PlCYP5 co-translationally interacted with the unassembled small ribosomal subunit protein, PlRPS15 (uS19). PlRPS15 contained an eukaryote-specific N-terminal extension that mediated the interaction with PlCYP5. PlCYP5 increased the solubility of PlRPS15 independent of its catalytic peptide-prolyl isomerase function and supported the integration of PlRPS15 into pre-ribosomes. Consistently, the phenotypes of the PlCYP5 loss-of-function mutant were similar to those of the PlRPS15 knockdown mutant (e.g. growth and ribosome biogenesis defects). PlCYP5 homologs in Arabidopsis thaliana, Homo sapiens, Schizosaccharomyces pombe, Sclerotinia sclerotiorum, Botrytis cinerea and Metarhizium anisopliae were identified. Notably, PlCYP5-PlRPS15 homologs from three filamentous fungi interacted with each other but not those from other species. In summary, our data disclosed a unique dedicated chaperone system for RPs by cyclophilin in filamentous fungi.

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

Cited by 8 publications

References 80 publications

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

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

Paralog-dependent Specialization of Paf1C subunit, Ctr9, for Sex Chromosome Gene Regulation and Male Germline Differentiation in Drosophila

Cyclophilin acts as a ribosome biogenesis factor by chaperoning the ribosomal protein (PlRPS15) in filamentous fungi

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