GSBS Dissertations and Theses

ORCID ID

0000-0002-6073-7667

Publication Date

2018-08-15

Document Type

Doctoral Dissertation

Academic Program

Interdisciplinary Graduate Program

Department

RNA Therapeutics Institute

First Thesis Advisor

Phillip D. Zamore

Keywords

nuage, mitochondria, Drosophila, piRNA, Armitage

Abstract

In the Drosophilaovary, PIWI-interacting RNAs (piRNAs) suppress transposon expression, ensuring female fertility. PIWI proteins Aub and Ago3, loaded with ping-pong piRNAs and reside in perinuclear nuage granules, engage in reciprocal transposon transcript cleavage termed the ping-pong cycle. The other PIWI protein Piwi, loaded with phased piRNAs and resides in the nucleus, silences transposon transcriptionally. Ping-pong piRNAs are made through the ping-pong amplification loop by Aub and Ago3, whereas phased piRNAs are made through consecutive endonucleolytic cleavages that spread in 5′-to-3′ direction, presumably by Zucchini (Zuc), an endonuclease resides on the surface of mitochondria. The ping-pong and phasing biogenesis pathways are coupled genetically and molecularly. However, it is not known how such coupling is achieved at the mechanistic level.

We found that nuage and mitochondria are physically separate under the confocal and electron microscopy. Zuc interacts with other known phasing factors on the mitochondrial surface, including an RNA-binding ATPase Armitage (Armi). Relying on its ATPase activity, Armi avoids binding to genic mRNAs, instead binds to piRNA precursors engaged in ping-pong or phasing, and localizes to both nuage and mitochondria. Armi localization is dynamically regulated by the ping-pong and phasing pathways. In armiloss-of-function mutants, ping-pong still operates, but phasing is disrupted. Therefore, the coupling between ping-pong and phasing pathways can be explained by Armi shuttling between nuage and mitochondria. An Armi ATPase mutant retains the interactions with piRNA biogenesis factors and piRNA precursors, but is insufficient to support phasing, suggesting an additional role of the Armi ATPase activity in ribonucleoprotein complex (RNP) remodeling.

Our study suggests that the dynamic distribution of an RNA-binding ATPase serves to transfer piRNA precursors between distinct subcellular compartments. It furthers our understanding of the complex coordination between piRNA biogenesis pathways and may serve to guide future studies on the mitochondrial phase of piRNA biogenesis. A few important questions remain to be answered: what interactions or conformational changes need to happen on Armi for it to anchor at nuage or mitochondria? How does Armi remodel the phasing RNP? Why are ping-pong and phasing machineries separated, and why does phasing happen on mitochondria?

DOI

10.13028/2v5a-rs66

Rights and Permissions

Copyright is held by the author, with all rights reserved.

Available for download on Wednesday, August 14, 2019

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