2 min readScientists Characterize Enzymatic Identity of Crucial Player in Generating Genome-protecting piRNAs

Structural studies of mouse Zucchini protein (mZuc) including this crystal structure obtained by Joshua-Tor and Ipsaro, helped determine that it acts as a nuclease which functions to process primary piRNA transcripts into shorter fragments, a first step in piRNA biogenesis. Image: CSHL

Cold Spring Harbor, NY  – Scientists at Cold Spring Harbor Laboratory (CSHL) have made important progress in understanding the earliest steps in a process that protects animal genomes from potentially dangerous genetic elements called transposons.

If left unchecked, these genomic parasites can run rampant and cause sterility.

The laboratories of CSHL Professors Gregory J. Hannon and Leemor Joshua-Tor, both Investigators of the Howard Hughes Medical Institute, joined forces to scrutinize the process by which a class of genome defenders called PIWI-interacting RNAs (piRNAs) are generated in cells of the germline – eggs and sperm that bear an organism’s genetic inheritance and transmit this information to offspring.

piRNAs are small RNA molecules (26-31 nucleotides in length) that bind to a class of germline-specific proteins called PIWIs.  Together, they form a molecular silencing machine, called RISC (RNA-induced silencing complex) which targets genomic parasites with great efficiency. RISC prevents parasitic sequences from becoming activated, and thus prevents their subsequent amplification and reinsertion into germline genomes.  If piRNA control is lost or disturbed, random insertion of active mobile elements into germline genomes leads to mutations, gross chromosomal abnormalities, and ultimately sterility.

Yet piRNA biogenesis – the means by which these specialized molecular silencers are generated – has eluded scientists.  In experiments conducted by postdoctoral researchers Astrid D. Haase of the Hannon lab and Jonathan J. Ipsaro of the Joshua-Tor lab, a protein called Zucchini that was previously implicated in the biogenesis of piRNAs was studied.  The aim was to detail its precise role in the biogenesis process, which remained unclear.

In prior work in fruit flies, Haase, Hannon and others demonstrated that when Zucchini was not expressed, single-stranded RNAs that are the precursors for functional piRNAs accumulated in germline cells.  This suggested that Zucchini was essential in piRNA biogenesis, however the molecular underpinnings of its mechanism were still unclear.

“We knew from our work in Drosophila that Zucchini functions at the initial processing step, in which long piRNA precursor molecules are cut into shorter fragments,” explains Haase.  “If it is to work properly, this cutting requires at least one endonuclease, an enzyme with very specific characteristics.”

The team knew that Zucchini – and its analogue in mice, called mouse Zucchini (mZuc, previously called PLD6/MitoPLD) – belongs to an enzyme family called phosphodiesterases.  That family includes members that can either act as phospholipases, cleaving phospholipid molecules, or as nucleases, cutting nucleic acid chains.  It was suspected, but not known for certain, that fly as well as mouse Zucchini proteins were nucleases – enzymes that specialize in cleaving bonds between the nucleotide subunits of nucleic acids (DNA and RNA).

In a paper published online in the journal Nature, the team reports the results of biochemical assays which demonstrate that mZuc has no phospholipase activity and that it does in fact possess nuclease activity.  

Ipsaro and Joshua-Tor performed a structural analysis that bolstered this finding and elucidated the molecular basis by which mZuc acts.  Using X-ray crystallography – a method in which patterns formed by high-powered X-rays shot through a crystal of the molecule reveal its atomic structure – the scientists found that the shape of the binding site in the mZuc protein was consistent with its identity as a nuclease. The finding was supported by a structural comparison of various members of the diverse phosphodiesterase family.

“On the basis of this evidence we propose that the Zucchini protein is in fact a nuclease, which functions to process primary piRNA transcripts into shorter fragments,” a critical first step in the biogenesis of a functional piRNA, Ipsaro and Haase report. 

“The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis” appears online ahead of print October 14, 2012 in Nature.  The authors are: Jonathan J. Ipsaro, Astrid D. Haase, Simon R. Knott, Leemor Joshua-Tor and Gregory J. Hannon.

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