The RNA-Binding ATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria - PubMed (original) (raw)

The RNA-Binding ATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria

Daniel Tianfang Ge et al. Mol Cell. 2019.

Abstract

PIWI-interacting RNAs (piRNAs) silence transposons in Drosophila ovaries, ensuring female fertility. Two coupled pathways generate germline piRNAs: the ping-pong cycle, in which the PIWI proteins Aubergine and Ago3 increase the abundance of pre-existing piRNAs, and the phased piRNA pathway, which generates strings of tail-to-head piRNAs, one after another. Proteins acting in the ping-pong cycle localize to nuage, whereas phased piRNA production requires Zucchini, an endonuclease on the mitochondrial surface. Here, we report that Armitage (Armi), an RNA-binding ATPase localized to both nuage and mitochondria, links the ping-pong cycle to the phased piRNA pathway. Mutations that block phased piRNA production deplete Armi from nuage. Armi ATPase mutants cannot support phased piRNA production and inappropriately bind mRNA instead of piRNA precursors. We propose that Armi shuttles between nuage and mitochondria, feeding precursor piRNAs generated by Ago3 cleavage into the Zucchini-dependent production of Aubergine- and Piwi-bound piRNAs on the mitochondrial surface.

Keywords: ATP; DTME; RNA-seq; argonaute; helicase; immunoprecipitation; mass spectrometry; microscopy; mitochondrion; small RNA.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.. Armi localizes to both nuage and mitochondria, physically separate sites of piRNA biogenesis

(A) Immunofluorescence detection of Vasa, ATP5A and Aub in wild-type stage 3 egg chambers. Box plots show the fraction of Vasa signal overlapping with ATP5A or Aub. Box comprises the 25th percentile and the 75th percentile of data, with the median indicated; whiskers mark 1.5 × interquartile range. _p_-value was calculated using the two-tailed Wilcoxon matched-pairs signed rank test. Immunofluorescence of Zuc-3×FLAG or Armi to detect their colocalization with ATP5A or Vasa in wild-type stage 3 egg chambers. (B) Transmission electron microscopy image of a wild-type stage 3 egg chamber. See also Figure S1.

Figure 2.. Phased piRNA production promotes nuage localization of Armi

(A) Immunofluorescence co-detection of Armi and Aub, Ago3, Vasa, Zuc-3×FLAG or ATP5A in wild-type or zucH169Y stage 3 egg chambers or of Armi and Aub or Zuc-3×FLAG in minotaurz3-5967 stage 3 egg chambers. Box plots show the fraction of Aub signal overlapping with Armi in wild-type, zucH169Y or minotaurz3-5967 stage 3 egg chambers. Box comprises the 25th percentile and the 75th percentile of data, with the median indicated; whiskers mark 1.5 × interquartile range. _p_-value was calculated using the two-tailed Mann Whitney test. (B) Scatter plots of the abundance of transposon- or gene-mapping 5′ monophosphorylated RNA >150 nt co-immunoprecipitated with Armi or control in wild-type or zucH169Y mutant ovaries. See also Figure S2–S4.

Figure 3.. Armi interacts with pre-pre-piRNA during the nuage and mitochondrial phases of piRNA production

(A) Genomic nucleotide bias around the 5′ ends (nt position 1) of 5′ monophosphorylated antisense transposon RNA >150 nt co-immunoprecipitated with Armi. Each RNA 5′ end was weighted equally, ignoring abundance. (B) Distance on the same genomic strand from 5′ ends of PIWI protein-bound piRNAs to 5′ ends of 5′ monophosphorylated long RNAs co-immunoprecipitated with Armi or control in wild-type or zucH169Y mutant ovaries.

Figure 4.. ArmiK729A ATPase mutant fails to support phased piRNA production

(A) Predicted Armi helicase core superimposed on the human Upf1 helicase core (Cheng et al., 2007). The enlarged view of the Armi ATP-binding pocket shows amino acids surrounding the adenosine-5′-(β,γ-imido)triphosphate (“ANP”) and a magnesium ion present in the Upf1 structure. (B) Scatter plot of the abundance of transposon- or gene-mapping piRNAs from the indicated genotype. (C) Distance on the same genomic strand from 5′ ends of piRNAs uniquely mapping to 42AB to the 5′ ends of 5′ monophosphorylated RNA >150 nt from the indicated genotype. _Z_-scores indicate the significance of the 5′-to-5′ distance = 10 nt on opposite genomic strands (ping-pong) and the 3′-to-5′ distance = 1 nt on the same genomic strand (phasing) for piRNAs. (D) Scatter plot of the abundance of transposon- or gene-mapping RNA >150 nt or piRNAs from the indicated genotype. See also Figure S5.

Figure 5.. Armi ATPase activity enables correct substrate selection

(A) Scatter plot of the abundance of transposon- or gene-mapping 5′ monophosphorylated RNA >150 nt co-immunoprecipitated with transgenic FLAG-Myc-ArmiK729A, FLAG-Myc-ArmiDE862,863AA, FLAG-Myc-ArmiE863Q versus FLAG-Myc-Armi. (B) Immunofluorescence detection using anti-FLAG antibody of transgenic FLAG-Myc-Armi, FLAG-Myc-ArmiK729A, FLAG-Myc-ArmiDE862,863AA or FLAG-Myc-ArmiE863Q in armi mutant germ cells from stage 3 egg chambers. See also Figure S6.

Figure 6.. A model for the role of Armi in piRNA biogenesis.

In addition to the three PIWI proteins and Zuc, other piRNA factors interacting with Armi are shown in blue.

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The RNA-Binding ATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria - PubMed (original) (raw)