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The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis

Abstract

PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism to provide essential protection for germ-cell genomes against the activity of mobile genetic elements1. piRNA populations comprise a molecular definition of transposons, which permits them to distinguish transposons from host genes and selectively silence them. piRNAs can be generated in two distinct ways, forming either primary or secondary piRNAs. Primary piRNAs come from discrete genomic loci, termed piRNA clusters, and seem to be derived from long, single-stranded precursors2. The biogenesis of primary piRNAs involves at least two nucleolytic steps. An unknown enzyme cleaves piRNA cluster transcripts to generate monophosphorylated piRNA 5′ ends. piRNA 3′ ends are probably formed by exonucleolytic trimming, after a piRNA precursor is loaded into its PIWI partner1,3. Secondary piRNAs arise during the adaptive ‘ping-pong’ cycle, with their 5′ termini being formed by the activity of PIWIs themselves2,4. A number of proteins have been implicated genetically in primary piRNA biogenesis. One of these, Drosophila melanogaster Zucchini, is a member of the phospholipase-D family of phosphodiesterases, which includes both phospholipases and nucleases5,6,7. Here we produced a dimeric, soluble fragment of the mouse Zucchini homologue (mZuc; also known as PLD6) and show that it possesses single-strand-specific nuclease activity. A crystal structure of mZuc at 1.75 Å resolution indicates greater architectural similarity to phospholipase-D family nucleases than to phospholipases. Together, our data suggest that the Zucchini proteins act in primary piRNA biogenesis as nucleases, perhaps generating the 5′ ends of primary piRNAs.

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Figure 1: mZuc acts as a nuclease but not a phospholipase in vitro.
Figure 2: mZuc acts as a single-strand-specific endoribonuclease in vitro.
Figure 3: Crystal structure of mZuc.
Figure 4: Electrostatic surfaces of PLD-family proteins indicate distinct binding surfaces for specific substrates.

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Primary accessions

Protein Data Bank

Data deposits

Coordinates were deposited in the Protein Data Bank under accession codes 4GGJ (native) and 4GGK (tungstate derivative).

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Acknowledgements

We thank W. Filipowicz, R. MacDonald and members of the G.J.H. and L.J. laboratories for discussions; G. Bencze, K. Rivera and D. Pappin of the Cold Spring Harbor Laboratory proteomics facility, which is funded in part by an National Cancer Institute Cancer Center Support Grant (CA045508), for support with mass spectrometry; and H. Robinson for help at the National Synchrotron Light Source, which is supported by the Department of Energy, Office of Basic Energy Sciences. J.J.I. was supported by the Harvey L. Karp award and by a Ruth L. Kirschstein National Research Service Awards National Institutes of Health (NIH) fellowship F32GM97888. This work was supported by NIH grant R01GM062534. G.J.H. and L.J. are Howard Hughes Medical Institute Investigators.

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L.J., G.J.H., A.D.H. and J.J.I. planned studies and wrote the paper. A.D.H. and J.J.I. performed the experiments, and S.R.K. analysed datasets.

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Correspondence to Leemor Joshua-Tor or Gregory J. Hannon.

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Ipsaro, J., Haase, A., Knott, S. et al. The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis. Nature 491, 279–283 (2012). https://doi.org/10.1038/nature11502

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