In vivo proximity labeling identifies the interactome of the endoribonuclease Zucchini in Drosophila ovary.

ABSTRACT: PIWI-interacting RNAs (piRNAs) are small RNAs that play a conserved role in genome defense by silencing transposable elements. The piRNA processing pathway is dependent on the sequestration of RNA precursors and protein factors in specific subcellular compartments. Therefore, a highly resolved spatial proteomics approach can help identify the local interactions and thereby elucidate the unknown aspects of piRNA biogenesis. Herein, we performed TurboID proximity labeling to investigate the interactome of Zucchini (Zuc), a key factor of piRNA biogenesis in germline cells and somatic follicle cells of the Drosophila ovary. Quantitative mass spectrometry analysis of biotinylated proteins defined the Zuc-proximal proteome, including the well-known partners of Zuc in piRNA biogenesis. Many of these were enriched in the cellular compartment of mitochondria or the outer mitochondrial membrane (OMM), where Zuc was specifically localized. A unique subset of proteins in the Zuc proximal proteome was characterized in comparison with the Tom20 proximal proteome, despite the overlapping subcellular localization of Zuc and Tom20 on OMM, indicating that the proximal proteomes in this analysis have target protein specificity beyond the subcellular localization dependency. Interestingly, the data indicated that chaperone function-related and endomembrane system/vesicle transport proteins are novel interacting partners of Zuc. The functional relevance of several candidates in piRNA biogenesis was validated by derepression of transposable elements after knockdown. Our results not only present potential Zuc-interacting proteins, but also suggest unrecognized biological processes, providing new insights into the physiological functions and molecular mechanisms of Zuc.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Ovary, Somatic Cell

SUBMITTER: Jong-Seo Kim  

LAB HEAD: Jong-Seo Kim

PROVIDER: PXD036182 | Pride | 2023-02-08

REPOSITORIES: Pride