The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis - PubMed (original) (raw)

. 2012 Nov 8;491(7423):279-83.

doi: 10.1038/nature11502. Epub 2012 Oct 14.

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

Jonathan J Ipsaro et al. Nature. 2012.

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 elements. 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 precursors. 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 partner. Secondary piRNAs arise during the adaptive 'ping-pong' cycle, with their 5' termini being formed by the activity of PIWIs themselves. 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 nucleases. 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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Figures

Figure 1

Figure 1. mZuc acts as a nuclease but not a phospholipase in vitro

a) The domain architecture of mZuc and Zuc are similar with an N-terminal transmembrane helix (TM, red) and a cytoplasmic domain, which contains the catalytic HKD motif (gold). The construct used for crystallization is indicated as a dashed box. Residue numbers delineating each domain indicated below each schematic. b) Phosphodiesterase activity for mZuc, its catalytic mutant (H153N), and a known phospholipase (Phospholipase D from Streptomyces chromofuscus, scPLD) were monitored by SRM-MS. Levels of the cardiolipin substrate and the expected phophatidic acid product (PA) are shown for each reaction. Error bars indicate standard deviation (n=3). c) DNase activity of recombinant mZuc was monitored using 50 nt 5'-32P-labeled DNA (red). Single-stranded (ss), double-stranded (ds), and partially double-stranded substrates were incubated with wild-type (wt) or catalytic mutant H153N enzyme (as indicated), and the resulting products separated by urea-PAGE. d) mZuc or the H153N mutant were incubated in the presence of 32PO43- followed by SDS-PAGE and transfer to a nitrocellulose membrane. e) The DNase of mZuc was measured in the presence of Na3VO4. scPLD is shown as control.

Figure 2

Figure 2. mZuc acts as a single-strand specific endoribonuclease in vitro

a) mZuc was incubated with ss, ds, and partially dsRNA substrates in the absence and presence of Na3VO4 as indicated. b) mZuc RNA cleavage products (from reactions shown in panel a, as indicated below) were tested for sensitivity to β-elimination and accessibility for polyadenylation.

Figure 3

Figure 3. Crystal structure of mZuc

a) The overall structure of the mZuc dimer is shown as a ribbon diagram. Helices are in green, strands in blue, and loops in beige. Each monomer binds one Zn2+ (yellow) in an extended zinc wing. The active site histidine residues (His153) are highlighted in red. b) A close-up of the zinc wing consisting of residues Cys49, Cys66, Cys68, and His72 is shown. c) A detailed view of mZuc co-crystallized with tungstate bound in the active site is presented.

Figure 4

Figure 4. Electrostatic surfaces of PLD family proteins indicate distinct binding surfaces for specific substrates

a) The electrostatic surface for mZuc displays a long, narrow, positively-charged grove laying across the zinc wings and active site. A short RNA molecule was manually built into the structure of mZuc, then subjected to energy minimization using GROMACS. The minimized model shows the phosphates of the RNA backbone positioned in the most positively charged areas of the groove with the bases extending away from the dimer core. b) In Nuc (PDB ID 1BYR), which acts on double-stranded DNA, the equivalent groove is significantly wider. c) A bone fide phospholipase, PLD (PDB ID 2ZE9), uses the same active site architecture in a strikingly different structural context. PLD has a small pocket for binding phospholipids, rather than an elongated groove. Each surface depicts the solvent-accessible surface contoured at ± 2 kB T/e using ABPS.

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