research highlightsnpg© 2012 Nature America, Inc. All rights reserved.

Zucchini has bite
The protein Zucchini belongs to the phospho­
lipase D (PLD) family of phosphodiesterases and has been
implicated in promoting
primary PIWI-interacting
RNA (piRNA) biogenesis.
PLD family members
include both phospho­
lipases and nucleases, but
it has been unclear whether
Zucchini has an impact on the piRNA biogenesis pathway indirectly(through a putative phospholipase activity) or directly (as a putativenuclease). Three recent reports address this question by ­analyzing
mouse and Drosophila melanogaster Zucchini (mZuc and DmZuc,respectively) using structural and functional approaches. Ipsaro et al.
determined the crystal structure of N-terminally truncated mZuc,
whereas Nishimasu et al. and Voigt et al. solved the structure of anequivalent truncation of DmZuc. The papers demonstrate that Zucchiniforms a dimer, with the active site assembled from conserved residues ofboth monomers, consistent with known monomeric and dimeric PLD
protein structures. The mZuc structure contains an unexpected CCCHtype zinc-finger motif, which has been implicated in the binding ofsingle-stranded RNA molecules, whereas DmZuc has a CHCC-typezinc-finger motif. In addition, the active site forms a narrow groove that
can accommodate single-stranded, but not double-stranded, nucleic
acids. Ipsaro et al. modeled a short RNA molecule into the structure ofmZuc, illustrating the shape and charge complementarity provided by
the RNA substrate. In support of the structural data, in vitro analyses­ hospholipase
confirmed that mZuc and DmZuc lacked any detectable pactivity but instead demonstrated single-strand–specific nuclease­activity. Nishimasu et al. further validated the functional ­significanceof DmZuc’s nuclease activity by demonstrating that wild-type dimeric
DmZuc is critical for transposon silencing. mZuc and DmZuc ­cleavage
generates 5′ phosphate and 3′ hydroxyl termini, which suggests thatZucchini might generate the 5′ ends of mature primary piRNAs,
which are known to bear a 5′-monophosphate group. Future studies
will be needed to pinpoint Zucchini’s precise role in primary piRNAb­ iogenesis. (Nature doi:10.1038/nature11502 and 10.1038/nature11509,
published online 14 October 2012; RNA doi:10.1261/rna.034967.112,
published online 19 October 2012)
AH

Pause-itively engaged
RNA polymerase II (Pol II) pausing downstream of gene p­ romoters
has emerged as a widespread phenomenon in eukaryotic gene
expression. Pausing could prime signal-responsive genes for­rapid activation; ­alternatively, it might serve as a checkpoint thatallows recruitment of the RNA-processing machinery. Although
the phenomenon—­originally described 20 years ago in Drosophila­melanogaster heat-shock genes—has gained acceptance as a core­element of transcription in higher eukaryotes, the status of Pol II at
the pause site has remained ­ambiguous. In a recent study, Core et al.
address the transcriptional status of p
­ romoter-associated Pol II byusing global run-on sequencing (GRO-seq). This method, which
relies on the detection of nascent RNA, allows precise ­determinationof the position, amount and orientation of transcriptionally engaged

polymerases genome wide. By p
­ roducing matched GRO-seq data setsin the presence or absence of Sarkosyl and quantitatively ­comparingthose to Pol II ChIP-chip and ChIP-seq results, the authors c­ onfirm
that a high degree of stable pausing occurs near promoters andshow that most, if not all, of the promoter-­associated polymerase
is in a transcriptionally engaged state rather than being part of a­postrecruitment preinitiation complex. In conjunction with s­ everal
earlier studies, this work cements the dynamic state of the paused­polymerase complex as a key tunable element of gene ­expression­regulation. (Cell Rep. doi:10.1016/j.celrep.2012.08.034, published
online 25 October 2012)
SLA PrePPI way to make predictions
The construction of protein-protein interactomes is an
important step toward understanding cell developmental
and regulatory programs. Protein-protein interactions (PPIs)
have been examined by using high-throughput experimentaltechniques, such as yeast two-hybrid assays and affinity
purification, and by computation-based approaches,
including gene ontology and phylogenetic profiles. Now,
Zhang et al. have developed an approach called PrePPI(predicting protein-protein interactions) that combines
structural- and nonstructural-interaction data to predict PPIs
on a genome-wide scale. PrePPI performs structural modelingon a pair of query proteins, making predictions based on close
structural neighbors as well as homology models and remote
structural relationships, greatly expanding the number ofinteractions that can be modeled. The resulting PPI modelis scored and, by using a Bayesian network, a likelihood
ratio is calculated that incorporates nonstructural evidence,such as coexpression levels and functional similarities.
The authors used PrePPI to score all possible pairwiseinteractions in the yeast and human proteomes, predicting
about 31,400 high-confidence interactions for yeast andnearly 318,000 for humans. Nineteen predictions wereexperimentally validated by using coimmunoprecipitation
assays. One set of predictions involved potential PPIs formedby suppressor of cytokine signaling 3 (SOCS3). SOCS3’s
inhibitory function had already been established in the JAKSTAT pathway, but PrePPI predicted interactions with GRB2
and RAG3, components of the RAS-MAPK pathway, whichwere experimentally confirmed. PrePPI performs better thanmethods that rely on structural or nonstructural evidence
alone. The authors attribute its relative success to the useof both close and remote structural neighbors, an efficientscoring strategy for their interaction models that allows themto examine a very large number of models, and the use ofa Bayesian network to evaluate the predicted interaction
models. Because PrePPI relies on structural representativesof sequence families, it falls short in mapping membraneprotein interactions. It also presently does not take posttranslational modifications into account. However, PrePPI
offers a feasible alternative to high-throughput methodologies
while also providing a rough model for what a predicted PPI
may look like. (Nature doi:10.1038/nature11503, publishedMMonline 30 September 2012)

Written by Arianne Heinrichs, Stéphane Larochelle & Michelle Montoya

nature structural & molecular biology volume 19 number 11 NOVEMBER 2012

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