Fernández-Vizarra, E., Tiranti, V., and Zeviani, M.
(2009). Biochim. Biophys. Acta 1793, 200–211.
Hällberg, B.M., and Larsson, N.G. (2014). CellMetab. 20, 226–240.
Herrmann, J.M., Woellhaf, M.W., and Bonnefoy, N.
(2013). Biochim. Biophys. Acta 1833, 286–294.
Li, G.W., Burkhardt, D., Gross, C., and Weissman,J.S. (2014). Cell 157, 624–635.

Mick, D.U., Dennerlein, S., Wiese, H., Reinhold,R., Pacheu-Grau, D., Lorenzi, I., Sasarman, F.,
Weraarpachai, W., Shoubridge, E.A., Warscheid,B., and Rehling, P. (2012). Cell 151, 1528–1541.
Richter-Dennerlein, R., Oeljeklaus, S., Lorenzi, I.,
Ronsör, C., Bareth, B., Schendzielorz, A.B.,
Wang, C., Warscheid, B., Rehling, P., and Dennerlein, S. (2016). Cell 167, this issue, 471–483.

Soto, I.C., Fontanesi, F., Liu, J., and Barrientos, A.
(2012). Biochim. Biophys. Acta 1817, 883–897.
Weraarpachai, W., Antonicka, H., Sasarman, F.,
Seeger, J., Schrank, B., Kolesar, J.E., Lochmüller,H., Chevrette, M., Kaufman, B.A., Horvath, R., andShoubridge, E.A. (2009). Nat. Genet. 41, 833–837.
Zhang, X., Zuo, X., Yang, B., Li, Z., Xue, Y., Zhou,Y., Huang, J., Zhao, X., Zhou, J., Yan, Y., et al.
(2014). Cell 158, 607–619.

PIWI Takes a Giant Step
Yibei Xiao1 and Ailong Ke1,*1Department of Molecular Biology and Genetics, Cornell University, 251 Biotechnology Building, Ithaca, NY 14853, USA*Correspondence: ailong.ke@cornell.edu
http://dx.doi.org/10.1016/j.cell.2016.09.043

piRNA guides the action of PIWI proteins to silence deleterious transposons in animal reproductivetissues. Biogenesis of piRNA-induced silencing complex (piRISC) involves a multi-step process. In
this issue, Matsumoto et al. report the first crystal structure of a PIWI-clade protein displaying aguide RNA, ready for action.
Argonaute (Ago) proteins play essential
roles in small-RNA-guided processes, bysilencing single-stranded (ss)RNAs complementary to its pre-loaded guide RNA(Swarts et al., 2014). They can be classified into two clades, AGO and PIWI. TheAGO-clade proteins are broadly distributed and mainly interact with 21–24nucleotide (nt) microRNAs (miRNAs) or
short interfering RNAs (siRNAs). Variantsof prokaryotic Agos may target DNA, using either RNA or DNA guides (Swartset al., 2014). The PIWI-clade proteins
are narrowly expressed in animal reproductive cells and associate with 26–
30 nt PIWI-interacting (pi)RNAs to form
piRNA-induced silencing complexes (piRISCs). PIWI-piRISCs protect animal germline cells by silencing deleterious transposons through either post-transcriptional
degradation or transcriptional silencing(Czech and Hannon, 2016).
The biogenesis of piRNA and theassembly of PIWI-piRISC are a rather
complicated process, which does not
involve a double-stranded (ds)RNA intermediate as in miRNA and siRNA pathways (Czech and Hannon, 2016). Instead,the long clustered piRNA transcript isendonucleolytically cleaved, and theintermediates are loaded onto PIWI andundergo further 30 end maturation, followed by 20 -O-methylation (20 -OME) atthe 30 terminal nucleotide (Iwasaki et al.,2015; Izumi et al., 2016) (Figure 1A).
Furthermore, a unique ping-pong amplification cycle serves as a secondary piRNAbiogenesis pathway, in which a subsetof piRNAs is selectively amplified upon
encountering transposon targets (Czechand Hannon, 2016) (Figure 1B). Studiesof piRNAi have been lagging behindbecause the pathway is restricted to animal gonads, and the specifics of piRNAbiogenesis and ping-pong mechanism
vary to some extent among differentsystems.
Structural studies on AGO-clade proteins have generated tremendous mechanistic insights. For example, key functional states for human (h)Ago2 with orwithout the miRNA guide, and with thetarget RNA, provide the structural basisfor understanding miRNAi (Schirle et al.,2014). However, similar effort on PIWIclade Argonautes was unsuccessful. In
recent years, several mechanistic breakthroughs have stemmed from studies

310 Cell 167, October 6, 2016 ª 2016 Elsevier Inc.

based on the silkworm Bombyx moriovary-derived BmN4 cells, which express
two PIWI proteins, Siwi and BmAgo3 (Kawaoka et al., 2008). In this issue of Cell,
Matsumoto et al. (2016) unveil the crystal
structure of the silkworm Siwi-piRISC
complex. The success doesn’t comeeasy, as it involves raising a monoclonal
antibody to affinity purify Siwi-piRISC
directly from BmN4 cells and using a protease to release the PIWI region for structure determination. The payoff, however,is well worth the effort. The authors note
structural differences of Siwi with theAGO-clade relatives, reveal piRNA recognition features, and examine the slicing
mechanism. These observations lay asolid foundation for more sophisticatedmechanistic dissections in the future.
Not surprisingly, Siwi bears a strong
structural resemblance to its AGO-clade
relatives. The four signature domains(N, PAZ, MID, and PIWI) and three linkers(L0, L1, and L2) assemble into a typicalbi-lobed (N-PAZ lobe and MID-PIWIlobe) architecture found in all Ago proteins(Figure 1C). However, the relative orientation of these two lobes differs significantly between Siwi and the AGO-cladeFigure 1. Schematic Diagram of piRNAi in Silkworm Ovary-Derived BmN4 Cells(A) The primary piRNA biogenesis pathway. After nuclease Zuccini-mediated cleavage of the clustered piRNA transcript, the piRNA intermediates are loaded onto
Siwi with the assistance of BmSpn-E/Qin. The subsequent binding of Papi and nuclease Trimmer defined the 30 end of piRNA, and Hen1 further catalyzes the20 -OH methylation (20 -OME) at the 30 terminal nucleotide.
(B) Ping-pong amplification cycle as the secondary piRNA biogenesis pathway. When piRNA guide encounters transposon RNA target, Siwi slices at the tenth
nucleotide, and nuclease BmVasa dissociates the cleavage products from Siwi. The 30 half of the product is then loaded onto Ago3 through g10A recognition,matured therein, and guides the slicing of piRNA transcripts to specifically generate more parental piRNA guides.
(C) Siwi-piRISCs’ (piRNA-induced silencing complexes) structure in a nutshell. Siwi assembles into a typical bi-lobed (N-PAZ lobe and MID-PIWI lobe) Agoarchitecture. It uses a tightly bound Mg2+ ion as part of the mechanism to read out the 50 phosphate (50 -PO4), and an aromatic pocket to accommodate 20 -OME
modification at the 30 end. The strong preferences for a uridine at the 50 end of guide RNA (g1U) and a complementary adenosine in the target RNA (t1A) are
explained based on protein-mediated contacts. An ‘‘unplugged’’ catalytic tetrad is found in the PIWI domain of Siwi-piRISC. Details in target RNA recognition,cleavage, and release await further structural snapshots.

representative, hAgo2. The N-PAZ lobe in
Siwi rotates significantly downward relative to its counterpart in hAgo2. Additional
hinge motions between N and PAZ domains exist, due to a distinct set of contacts at the interface, which includes
an AGO-clade-specific insertion. SiwipiRISC has a robust slicing activity. Theauthors point out that three out of thefour residues in the DEDH catalytic tetrad
are well-preserved in 3D in the RNase
H-fold of PIWI domain. The fourth residue,a glutamate finger in the disordered loop 2of PIWI domain, likely joins the active
site upon the guide-target RNA duplex
formation. This ‘‘unplugged’’ active site
scheme is found in Agos of some bacteria. By contrast, eukaryotic Agos containa ‘‘plugged-in’’ slicer active site, in which

all four residues in the catalytic triad are
pre-organized.
PIWI proteins prefer a much longer RNA
guide, containing a phosphate at the 50end and a 20 -OME modification at the30 end. Drosophila Aubergine, SIWI, andmany other PIWI proteins strongly specify
a uridine in the first position of their guide(g1U preference) and a complementary
adenosine in the target (t1A bias). Theirping-pong cycle partner, Ago3, specifies
g10A in its guide, which is equivalent
to t1A before PIWI slicing; therefore, theslicing product of one naturally serves as
the guide for the other, triggering an inphase amplification cycle. In the SiwipiRICS structure, even though the middle
portion of the 28 nt endogenous
piRNA is disordered, recognitions to thesequence and chemical features at thepiRNA ends are crystal clear. Siwi uses
a tightly bound Mg2+ ion as part of themechanism to read out the 50 phosphate—again, a scheme not found in eukaryotic Ago, but popular among prokaryotic ones. The g1U preference is nicelyexplained, based on the observation thata specificity loop in Siwi MID domain flips
g1U into a tightly surrounded pocket andaccepts a hydrogen bond from N3 of
g1U for sequence readout. A and G are
too big to fit into the pocket, and C does
not satisfy the hydrogen bonding pattern.
As for the t1A bias, the structure supports
the conclusion that t1A is specified
through protein-mediated recognition,rather than base-pairing readout (Wanget al., 2014). Following g1U, SIWI
Cell 167, October 6, 2016 311

presents a stretch of four nucleotides(g2–g5) in pseudo-A form conformation.
This so-called ‘‘seed sequence region’’is much shorter than that presented bythe AGO-clade proteins, hence the target
searching mechanism may be somewhat
different. The mechanism to accommodate 20 -OME at the 30 end of piRNAagrees with the observations from adomain structure (Tian et al., 2011). Interactions involve a set of tyrosine residues
hydrogen bonding with the 30 -OH and20 -oxygen of the terminal nucleotide, as
well as an aromatic pocket accommodating the methyl modification.
The preference for a longer guide RNA
can also be rationalized to some extent.
Whereas miRNA is found to travel between the MID-PIWI and N-PAZ lobes in
hAgo2, no RNA densities are found in
the equivalent region of Siwi-RISC. Significant slack must be present in the Siwibound piRNA. This is likely caused by
a PIWI-clade-specific insertion into thePAZ domain, which forces piRNA outward to travel a longer path. Based on
the observed MID-PIWI and N-PAZ orientation differences, the authors further
speculate that the formation of theguide-target RNA duplex would cause
less geometry distortion in Siwi than inhAgo2, hence the sliced product is underless pressure to release autonomously,
which explains the requirement of helicases such as BmVasa in piRISC product

312 Cell 167, October 6, 2016

release. Both speculations would require
additional work to solidify into reliablemechanisms, by replacing structuralmodels with real snapshots of SiwipiRISC loaded with a homogeneouspiRNA guide and programmed into theguide-target RNA duplex stage, before
and after the slicing action.
In summary, the Siwi-piRICS structure
ascends the mechanistic discussion ofpiRNAi to a higher level. Many cellular factors influence the biogenesis and functional outcome of piRISC through direct
or indirect interactions with PIWI proteins.
For example, in the silkworm system,
nuclease Trimmer and adaptor protein
Papi interact with Siwi proteins containing
an sDMA modification. RNA cleavage
takes place inside this ternary complex
to define the 30 end of piRNA (Izumiet al., 2016). piRNA must have traveled adifferent path from observed in the SiwipiRISC structure, and the measuring
mechanism inside this complex may wellbe the major determinant for piRNA length
preference. Similarly, BmSpn-E/BmQin
dimer was found to assist primary piRNA
processing by interacting with Siwi,
whereas a germline-specific DEAD box
RNA helicase BmVasa forms complex
with Siwi to facilitate the release of thesliced target RNAs from Siwi-piRISC,
ensuring efficient ping-pong cycling(Nishida et al., 2015). piRISC exists inthese two mutually exclusive complexes

during different stages of its function cycle. Future structure and function dissections of these higher-order molecular assemblies will no doubt generate valuable
insights into the piRNAi pathway.
REFERENCES
Czech, B., and Hannon, G.J. (2016). Trends Biochem. Sci. 41, 324–337.
Iwasaki, Y.W., Siomi, M.C., and Siomi, H. (2015).
Annu. Rev. Biochem. 84, 405–433.
Izumi, N., Shoji, K., Sakaguchi, Y., Honda, S.,Kirino, Y., Suzuki, T., Katsuma, S., and Tomari, Y.
(2016). Cell 164, 962–973.
Kawaoka, S., Hayashi, N., Katsuma, S., Kishino,H., Kohara, Y., Mita, K., and Shimada, T. (2008).
Insect Biochem. Mol. Biol. 38, 1058–1065.
Matsumoto, N., Nishimasu, H., Sakakibara, K.,
Nishida, K.M., Hirano, T., Ishitani, R., Siomi, H.,Siomi, M.C., and Nureki, O. (2016). Cell 167, thisissue, 484–497.
Nishida, K.M., Iwasaki, Y.W., Murota, Y., Nagao,A., Mannen, T., Kato, Y., Siomi, H., and Siomi,M.C. (2015). Cell Rep. 10, 193–203.
Schirle, N.T., Sheu-Gruttadauria, J., and MacRae,I.J. (2014). Science 346, 608–613.
Swarts, D.C., Makarova, K., Wang, Y., Nakanishi,K., Ketting, R.F., Koonin, E.V., Patel, D.J., andvan der Oost, J. (2014). Nat. Struct. Mol. Biol. 21,743–753.
Tian, Y., Simanshu, D.K., Ma, J.B., and Patel, D.J.
(2011). Proc. Natl. Acad. Sci. USA 108, 903–910.
Wang, W., Yoshikawa, M., Han, B.W., Izumi, N.,Tomari, Y., Weng, Z., and Zamore, P.D. (2014).
Mol. Cell 56, 708–716.