DEAD‐box polypeptide 43 facilitates piRNA amplification by actively liberating RNA from Ago3‐piRISC

Murakami, Ryo; Sumiyoshi, Tetsutaro; Negishi, Lumi; Siomi, Mikiko C.

doi:10.15252/embr.202051313

Cited by 15 publications

(35 citation statements)

References 47 publications

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“…In vivo, product release has been proposed to be facilitated in insects and mammals by the RNAstimulated ATPase Vasa (DDX4) [88][89][90] and the Vasa-like protein DDX43 (ref. 91).…”

Section: Gtsf1 Is Necessary and Sufficient For Efficient Target Cleav...mentioning

confidence: 99%

The conserved zinc-finger protein GTSF1 helps PIWI proteins achieve their full catalytic potential

Arif

Özata

Anderson

et al. 2021

Preprint

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Argonaute proteins use nucleic acid guides to find and bind specific DNA or RNA target sequences. Argonaute proteins can be found in all kingdoms of life, and play diverse biological functions including genome defense, gene regulation, and chromosome partitioning. Many Argonautes retain their ancestral endoribonuclease activity, cleaving the phosphodiester bond between target nucleotides t10 and t11. In animals, a specialized class of Argonautes, the PIWI proteins, use 21–35 nt PIWI-interacting RNAs (piRNAs) to direct transposon silencing, protect the germline genome, and regulate gene expression during gametogenesis1. The piRNA pathway is required for fertility in one or both sexes of nearly all animals. Both piRNA production and function require RNA cleavage catalyzed by PIWI proteins. Spermatogenesis in mice and other placental mammals requires three distinct, developmentally regulated PIWI proteins: MIWI (PIWIL1), MILI (PIWIL2), and MIWI2 (PIWIL4)2 4; the piRNA-guided endoribonuclease activities of MIWI and MILI are essential to produce functional sperm5,6. piRNA-directed silencing in mice, insects, and worms also requires Gametocyte-Specific Factor 1 (GTSF1), a PIWI-associated protein of unknown function7 12. Here, we report that GTSF1 potentiates the weak, intrinsic, piRNA-directed RNA cleavage activities of PIWI proteins, transforming them into efficient endoribonucleases. GTSF1 represents the first example of an auxiliary protein that potentiates the catalytic activity of an Argonaute protein.

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“…In vivo, product release has been proposed to be facilitated in insects and mammals by the RNAstimulated ATPase Vasa (DDX4) [88][89][90] and the Vasa-like protein DDX43 (ref. 91).…”

Section: Gtsf1 Is Necessary and Sufficient For Efficient Target Cleav...mentioning

confidence: 99%

The conserved zinc-finger protein GTSF1 helps PIWI proteins achieve their full catalytic potential

Arif

Özata

Anderson

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…Finally, in the ping-pong model, DDX43, one of DEAD-box RNA helicase recently identified, was reported to dissociate truncated RNA from Ago3-piRISC 10 . In contrast, Vasa is responsible for dissociating RNA from Siwi-piRISC.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, germ cells have an ingenious piRNA amplification mechanism for efficient transposon RNA cleavage, known as the ping-pong pathway 9 . In this pathway, Siwi, Vasa, Ago3, and DDX43 are orchestrate a droplet called Nuage near the nuclear membrane in silkworm germ cells BmN4 8 , 10 .…”

Section: Introductionmentioning

confidence: 99%

Heterogeneous dissociation process of truncated RNAs by oligomerized Vasa helicase

et al. 2021

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RNA helicases are enzymes that generally unwind double-stranded RNA using ATP hydrolysis energy, mainly involved in RNA metabolism, transcription, translation, and mRNA splicing. While the helicase core is crucial for RNA unwinding activity, N- and C-terminal extensions of specific helicases may contain an intrinsically disordered region for electrostatic interaction, resulting in the formation of droplets in the cytoplasm. However, how the disordered region of the RNA helicase contributes to RNA unwinding and dissociation remains unclear. Here, we focused on Bombyx mori Vasa, which unwinds truncated target transposon RNAs from the piRNA-induced silencing complex piRISC. In this study, we used single-molecule techniques to visualise how Vasa dynamically interacts with piRISC and investigate how Vasa oligomerization is involved in the process of piRNA amplification, named the ping-pong pathway. We found that Vasa’s oligomerization is required during these processes in vitro and in vivo, and that Vasa triggers the dissociation of truncated RNA in heterogeneous pathways. Our single-molecule results suggest that oligomerized Vasa guides the timing of the process regulating overall dissociation efficiency.

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“…In concert with the robust production of post-meiotic round spermatids, these results suggest that DDX43 has no major influence on meiotic progression. DDX43 was recently reported to function as a Vasa-like protein to facilitate amplification of Piwi-interacting RNAs (piRNAs) in an ovary-derived cell line of silkworms 60 . In mammalian testis, pachytene piRNAs, as one predominant class of piRNAs, are generated in pachytene spermatocytes and round spermatids 61,62 on its expression pattern, DDX43 might regulate pachytene piRNA biogenesis in mouse testis.…”

Section: Ddx43 Deficiency Is Insufficient To Frustrate Meiosis and Pa...mentioning

confidence: 99%

Single-Cell RNA-seq Uncovers Dynamic Processes Orchestrated by RNA-Binding Protein DDX43 in Chromatin Remodeling during Spermiogenesis

Tan

Zhou

Wang

et al. 2022

Preprint

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Advances in single-cell RNA sequencing (scRNA-seq) have allowed for elucidating biological mechanisms at cell state level. Mammalian spermatogenic process showcases dynamic switches of gene expression pattern with delicate morphological and functional alterations of germ cells, but it is unclear how such dynamics is genetically controlled. Here we demonstrate that mouse testis-enriched RNA helicase DDX43, as well as its ATP hydrolysis site, is required for spermiogenesis. Genetic mutation of Ddx43 renders spermatids heterogeneously defective in multiple steps of chromatin remodeling, resulting in incomplete substitution of transition protein by protamine and less condensed sperm nucleus. Through scRNA-seq analyses of testicular cells derived from adult wild-type and Ddx43 mutant testes in mice, we reveal that the DDX43 deficiency-elicited perturbation in the dynamic RNA regulatory processes underlies the differentiation deficiency of spermatids. Further, focused analyses on early-stage spermatids combined with enhanced CLIP sequencing (eCLIP-seq) identify Elfn2 as DDX43-targeted hub gene, whose in vivo knockdownshows similar phenotypic defects as Ddx43 mutant. Our study illustrates an essential role for DDX43 in post-meiotic chromatin remodeling and highlights the single cell-based strategy for a refined dissection of stage-specific regulation of germline differentiation.

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DEAD‐box polypeptide 43 facilitates piRNA amplification by actively liberating RNA from Ago3‐piRISC

Cited by 15 publications

References 47 publications

The conserved zinc-finger protein GTSF1 helps PIWI proteins achieve their full catalytic potential

The conserved zinc-finger protein GTSF1 helps PIWI proteins achieve their full catalytic potential

Heterogeneous dissociation process of truncated RNAs by oligomerized Vasa helicase

Single-Cell RNA-seq Uncovers Dynamic Processes Orchestrated by RNA-Binding Protein DDX43 in Chromatin Remodeling during Spermiogenesis

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