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Siwi cooperates with Par-1 kinase to resolve the autoinhibitory effect of Papi for Siwi-piRISC biogenesis

Hiromi Yamada1, Kazumichi M Nishida1, Yuka W Iwasaki2,3

  • 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, 113-0032, Japan.

Nature Communications
|March 22, 2022
PubMed

Insights

Bombyx Papi scaffolds Siwi-piRISC biogenesis by binding piRNA precursors. Siwi kinase Par-1 phosphorylates Papi, enhancing RNA binding and facilitating piRISC assembly on mitochondria.

Area of Science:

  • Molecular Biology
  • RNA Biology
  • Biochemistry

Background:

  • Bombyx Papi protein is crucial for Siwi-piRISC biogenesis on mitochondria.
  • Papi binds Siwi and piRNA precursors, but the mechanism of RNA binding regulation is unknown.
  • Phosphorylation of Papi is essential for its interaction with piRNA precursors.

Purpose of the Study:

  • To elucidate the mechanism by which Papi phosphorylation regulates Siwi-piRISC biogenesis.
  • To identify the kinase responsible for Papi phosphorylation and its target site.
  • To understand the role of specific Papi domains in Siwi and RNA binding.

Main Methods:

  • Site-directed mutagenesis of Bombyx Papi (S547A mutant, deletion mutants).
  • Biochemical assays to assess protein-protein and protein-RNA interactions.
  • Analysis of Siwi-piRISC biogenesis in vitro and in vivo.

Main Results:

  • Siwi recruits Par-1 kinase to phosphorylate Papi at Ser547.
  • Phosphorylation at Ser547 enhances Papi's K-homology (KH) domain binding to Siwi-bound piRNA precursors.
  • Papi mutants lacking key domains exhibited defects in Siwi interaction, phosphorylation, or RNA binding, disrupting piRISC assembly.
  • A Papi mutant lacking Tudor and auxiliary domains showed autonomous RNA binding.

Conclusions:

  • Siwi and Par-1 kinase cooperate to regulate Papi phosphorylation, a critical step for Siwi-piRISC biogenesis.
  • Papi's auxiliary domain and Ser547 phosphorylation are essential for coordinated piRISC assembly.
  • This study reveals a sophisticated regulatory mechanism for piRISC formation on the mitochondrial surface.

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