-
The International Journal of Biochemistry & Cell Biology 42
(2010) 13341347
Contents lists available at ScienceDirect
The International Journal of Biochemistry& Cell Biology
journa l homepage: www.e lsev ier .com
Small R om
Nelson CDepartment of
a r t i c l
Article history:Available onlin
Keywords:piRNAmiRNAsiRNAgermlinePiwi
, pacryonicell dpathwmiRNmement,A acchanents.the fuals
ofents.
1. Introduction
Small RNAs and the RNAi pathway are key components of
generegulation systems in eukaryotic cells. In animals, the proper
devel-opment of sRNAi pathwdistinct frodevelopmeexhibit an ubecome
thetory RNAs.
MicroRNtwo promin(Fig. 1). Whger RNAs mand Sonthewith innate(TEs
or transomatic celcertain miRMurchisonHowever, tsiRNAs
(endgermline-sp
Tel.: +1 61E-mail add
interacting RNAs (piRNAs). Despite their abundance, piRNAs
andendo-siRNAs have only recently been unveiled, and many aspectsof
their molecular functions are still undened.
To date, nearly all small regulatory RNAs function in a
complex
1357-2725/$ doi:10.1016/j.omatic tissues is intimately dependent
on a functioningay (Ambros, 2004). Although germ cells are
uniquely
m somatic cells because they form gametes, germ cellnt also
appears to require RNAi. However, germ cellsnanticipated diversity
of RNAi mechanisms and haverichest environment for the discovery of
small regula-
As (miRNAs) and small interfering RNAs (siRNAs) areent classes
of small regulatory RNAs in eukaryotesile miRNAs regulate many
endogenous target messen-ainly through gene silencing (Bartel,
2009; Carthew
imer, 2009), siRNAs are thought to provide organismsdefenses
against viruses and transposable elementssposons). miRNAs were
largely rst characterized in
ls, but it was anticipated that germ cells would expressNAs
specic to the germline (Mishima et al., 2008;et al., 2007; Ro et
al., 2007; Watanabe et al., 2006).he surprises lurking in germ
cells were endogenouso-siRNAs) not previously seen in somatic
cells, and aecic, abundant class of longer small RNAs called
Piwi-
7 724 5455.ress: [email protected].
with an Argonaute (AGO) or PIWI protein. Most animal
genomescontain multiple homologs of these proteins. Each possesses
a PAZdomain, which binds the 3 end of small RNAs, and a PIWI
domain,which folds into an RNase H-like fold and can cleave a
target RNAstrand that is matched to the guide RNA bound in the
domain(Slicer activity, Tolia and Joshua-Tor, 2007). AGO proteins
pre-fer to bind2023nucleotide (nt) miRNAs and siRNAs, while
PIWIproteins prefer to bind2431nt long Piwi-interacting RNAs
(piR-NAs). miRNAs and siRNAs are processed by the Dicer
endonucleasefrom precursors with double-stranded RNA (dsRNA)
features; piR-NAs are distinct because they are typically longer in
length, theirprecursors, in general, lack dsRNA features, and their
productiondoes not require Dicer (Ghildiyal and Zamore, 2009).
Several recent reviews discuss the discovery of these
smallregulatory RNAs (Ghildiyal and Zamore, 2009; Malone andHannon,
2009; Klattenhoff and Theurkauf, 2008). The miRNA andsiRNA pathways
have also been recently reviewed (Carthew andSontheimer, 2009; Kim
et al., 2009; Okamura and Lai, 2008). There-fore, this
germline-centric review of small regulatory RNAs willfocus on the
two newest classes of germ cell small RNAs: piRNAsand endo-siRNAs.
I will discuss the history of how germline devel-opment studies
have now intersected with the biology of smallregulatory RNAs.
Finally, I will address the current challenges inunderstanding
small RNA biology within the context of germ cells
see front matter 2010 Elsevier Ltd. All rights
reserved.biocel.2010.03.005NAs in the animal gonad: Guarding
gen
. Lau
Biology, Brandeis University, 415 South Street, Waltham, MA
02454, USA
e i n f o
e 19 March 2010
a b s t r a c t
Germ cells must safeguard, apportionensure proper reproduction
and embmany genes controlling animal germlinked to the RNA
interference (RNAi)RNAs. Germ cells contain microRNAs (RNAs
(piRNAs); these are bound byknown to specify germ cell
developmdevelopment can also affect small RNsurprising diversity of
regulatory meling the spread of transposable elemgermline small
RNAs and to identifyarea will likely impact biomedical gothe
transposition of mobile DNA elem/ locate /b ioce l
es and guiding development
kage, and deliver their genomes with exquisite precision toc
development. Classical genetic approaches have identiedevelopment,
but only recently have some of these genes beenay, a gene
silencingmechanism centered on small regulatoryAs), endogenous
siRNAs (endo-siRNAs), and Piwi-interactingbers of the
Piwi/Argonaute protein family. piwi genes werebut we now understand
that mutations disrupting germlinecumulation. Small RNA studies in
germ cells have revealed aisms and a unifying function for germline
genes in control-Future challenges will be to understand the
production ofll breadth of gene regulation by these RNAs. Progress
in thismanipulating stem cells and preventing diseases caused
by
2010 Elsevier Ltd. All rights reserved.
-
N.C. Lau / The International Journal of Biochemistry & Cell
Biology 42 (2010) 13341347 1335
Fig. 1. Gonada ll interRNAs that are
and proposcations for
2. Where i
Our undanimals catode CaenordsRNA triggtal regulatoWightmanshown
thatbacteria explong after ttransmissioet al., 2000;
The Plastors that disthese mutasterility thawas carriedteinwas
amBoth the Mtor strains wwith dsRNAas rde-3, likwere
allelic1999).
In a diffegans mutanmutations iRNA polymgal RdRP gedefend
agaifound to berespondingother RdRP1, -2, and -3gene silenciRdRP
genesterk laboraRNAi respo2005; Simmeri-1 enhandependentmut-7.
Althouggermline de
ergina plnou
riggewasays iays ton limmedmal cthe
all RNNAsns begar2003of ne006)y RdRsphad Fiy thre dintiafurthans
gto e
and ionadolfswline
tinctroteihat ll small RNAs that aregeneratedbyDicer.MicroRNAs
(miRNAs) andendogenous smaincorporated into Argonaute (AGO)
proteins.
e that germ cell focused studies will have broad
impli-understanding animal health and evolution.
t began: RNAi and the nematode germline
erstanding of small RNA-mediated gene regulation inn be traced
to pioneering discoveries in the nema-habditis elegans (C.
elegans), such as the revelation thaters RNAi (Fire et al., 1998)
and that the developmen-r genes lin-4 and let-7 encode miRNAs (Lee
et al., 1993;et al., 1993; Reinhart et al., 2000). In C. elegans,
it wasRNAi can be induced simply by feeding the
nematodesressingdsRNA, and that this effectpersists intoprogenyhe
source of the dsRNA trigger is removed, suggestingn and amplication
of siRNAs in the germline (GrishokSijen et al., 2001; Timmons and
Fire, 1998).
terk laboratory isolatednematodemutants calledmuta-played
elevated mobilization of transposons; some ofnts, such as mut-7,
displayed a temperature-dependentt was most evident when the
homozygous mutationby the hermaphrodite, suggesting that the MUT-7
pro-aternally deposited gene product (Ketting et al., 1999).ello
and Plasterk laboratories found that some muta-ere unable to mount
an RNAi response when injected, while some RNAi-decient (rde)
mutant strains, suchewise displayed elevated transposon
mobilization andwith mutator genes (Ketting et al., 1999; Tabara et
al.,
rent approach, the Maine laboratory isolated a C. ele-t that was
severely defective in oogenesis and carriedn the gene ego-1, a
homolog of plant RNA-dependenterases (RdRP, Smardon et al., 2000).
Plant and fun-nes are necessary for mounting an RNAi response tonst
viruses (Chen, 2009),while in nematodes, ego-1was
an emnomenendogeRNAi tcells, itpathwpathwcommsiRNA-
Forbehindest smof miRC. elegaRNAs ret al.,cationet al., 2ated
btripho(Pak anstudy btions aprefere
ToC. elegappliedment,in thegvan Wof germare disAGO pRdRP
tnecessary for proper meiosis and mitosis, as well as forto dsRNA
to initiate RNAi. In addition to ego-1, threehomologs are encoded
in the C. elegans genome: rrf-, and mutations in these genes all
exhibit a dampenedng response (Smardonet al., 2000). Convergingon
theseas well as uncovering new genes, the Ruvkun and Plas-tories
discovered genes that enhance or suppress thense in somatic cells
(Kennedy et al., 2004; Kim et al.,er et al., 2002). For example,
mutations in rrf-3 and
ce RNAi in neurons, and curiously cause a temperature-sterility
defect, analogous to the phenotype seen in
h the links between RNAi, transposon regulation, andvelopment in
the nematode were not clear at the time,
certain mRmechanism
3. Not justendo-siRNA
Therstto C. eleganassumptionhigher animor RRF-1.
NsiRNAgenewithout anan aggressiferingRNAs (endo-siRNAs)
areprocessed intomature single-stranded
g hypothesis was that RNA-based gene silencing phe-ayed a role
in germ cell formation through putatives siRNAs and protein
factors. It was thought that whilered by exogenous dsRNAs was
robust in many somaticalsodampenedwhencompetingwithendogenousRNAin
the germline. Thus, mutations in endogenous RNAihat lowered
endo-siRNA levels were thought to liberate
iting RNAi factors to be used for effective exogenousiated
silencing.haracterization of endo-siRNAs from C. elegans
laggedbroader discovery of miRNAs, in part because the earli-A
cloning methods focused on the 5 monophosphate
and exogenous siRNAs. However, the endo-siRNAs inecame apparent
with cloning techniques that captureddless of the status of the
small RNAs 5 end (Ambros; Lee et al., 2006; Pak and Fire, 2007) and
in identi-w factors involved in small RNA biogenesis (Duchaine. An
examination of secondary siRNA molecules gener-Ps in nematodes
indicated that they contain 5 di- and
tes, andmight be unprimed RdRP products frommRNAsre, 2007; Sijen
et al., 2007). Finally, a deep sequencinge Bartel laboratory
suggested that endo-siRNA popula-verse in C. elegans, with 21-,
22-, and 26-nt species thatlly start with a 5 guanosine (Ruby et
al., 2006).er understand the repertoire of endo-siRNAs in
theermline, deep sequencing efforts have recently beenmbryos,
gametes, mutants affecting germline develop-mmunoprecipitates of
nematode AGO proteins residents (Claycombetal., 2009;Guetal.,
2009;Hanetal., 2009;inkel et al., 2009). These studies codied two
classesendo-siRNAs, 26G RNAs and 22G RNAs (Fig. 2), whichin their
length, the factors that generate them, and thens that bind them.
Both 26G and 22G RNAs require anikely synthesizes an unprimed
antisense transcript to
NAs expressed throughout the genome; however, thefor selecting
the specic mRNAs is currently unknown.
nematodes: insects and vertebrate gonadals
discoveredmiRNA, lin-4,was once thought to beuniques because of
lack of obvious animal homologs; thismay have also been extended to
endo-siRNAs becauseals lack an endogenous RdRP homologous to
EGO-1
evertheless, long dsRNAs that are substrates for endo-ration
canbe formedby single-strandedRNAprecursorsRdRP (Fig. 1). However,
most mammalian cells mountve innate immune response to
intracellular dsRNA that
-
1336 N.C. Lau / The International Journal of Biochemistry &
Cell Biology 42 (2010) 13341347
Fig. 2. Gonadawith the expanthis nematodebesidesmiRNA(piRNAs)
baseproteins of othin gonads, andinitial steps ofthe
predominacontain a 5 mphate. The mosiRNAs/piRNA
is indicative2007), withactivate the2000).
Using demouse oocynot only mstantial num2008). The mwhich the
mnumber canlarge invertsive complepseudogeneurations progenerate
encomplex (T
To validproled in d
scripts like Ran-GAP and Ppp4r1 were up-regulated 24 foldin
mutant oocytes. Ran-GAP is a protein implicated in control-ling the
molecular gradient of Ran-GTP, which mediates spindleorganization
(Kalab and Heald, 2008). The substantial number
o-siRns, lindleison
NAi cmain008)rtly aortshilaal eb; Ced noneidef embra
eo-siROkamrestiof endfunctiootic sp(Murchthat Rthis reet al., 2
Shople repDrosopGhildiy2008a,detector Schcells oOkamuy
end2009;
Intel small RNAs specic to the nematode Caenorhabditis elegans.
In lineded number of Argonaute-family proteins in the C. elegans
genome,s gonad also contains an additional layer of small
regulatory RNAss and siRNAs. 21URNAsmaybeorthologous to
Piwi-interactingRNAsd on the homology of Piwi-related gene-1 and -2
proteins to PIWIer animals. 26G and 22G RNAs are siRNAs most
abundantly detectedappear to require an RNA-dependent RNA
Polymerase (RdRP) forbiogenesis. These RNAs are named after their
signature length andnt 5 nucleotide in that class. While 21U and
26G RNAs likely oftenonophosphate, 22G RNAs predominantly contain a
5 di- or triphos-del here speculates that 26G and 21U RNAs might
serve as primarys that then promote generation of secondary siRNAs
(22G RNAs).
of a viral infection (interferon response, SenandSarkar,the
notable exception being germ cells, which do notresponse when
injected with dsRNA (Svoboda et al.,
ep sequencing of small RNAs from many thousands oftes, the
Hannon and Watanabe laboratories identiedany known miRNAs and some
piRNAs, but also a sub-ber of endo-siRNAs (Tam et al., 2008;
Watanabe et al.,ouse oocyte endo-siRNAs are mainly 2122nt long,
ofajority map to transposons; in addition, a signicantalso be
mapped to pseudogenes that either contain a
ed repeat (e.g. theRan-GAPpseudogene)or showexten-mentarity to
an endogenous gene (e.g. Ppp4r1 and its) (Fig. 1). The groups
postulated that these gene cong-duce dsRNA precursors that are
processed by Dicer todo-siRNAs that are loaded into theArgonaute-2
(Ago-2)am et al., 2008; Watanabe et al., 2008).ate a function for
oocyte endo-siRNAs, mRNAs wereicer-null and ago-2-null oocytes.
Predicted target tran-
cells map tomdg1, 297,Ghildiyal eaccount onling
TE-assoendo-siRNAet al., 2008siRNAsmapmagnitudesus siRNAsFor
examplblood are acorrespondal., 2008). Itranscriptio
4. A small
Studiessmall RNAsin animals;NAs) (Arav2005). ThesinteractingRNAs
co-pumals (Aravand Zamorbeen next-al., 2009), wof individuaalso
been dworm, sponet al., 2008et al., 2009yielded moorigins.
Piwi-intbased on wI piRNAs prmap uniqustrand bias1100kilobNAs
that target genes with microtubule-associatedike Ran-GAP, as well
as the pronounced defects in mei-
formation previously observed in dicer-null oocyteset al., 2007;
Tanget al., 2007), have led to the suggestionan modulate the
microtubule cytoskeleton, althoughs to be tested molecularly (Tam
et al., 2008; Watanabe.fter mouse oocyte endo-siRNAs were
described, multi-followed with deep sequencing of endo-siRNAs
frommelanogaster (D. melanogaster) (Czech et al., 2008;t al., 2008;
Kawamura et al., 2008; Okamura et al.,hung et al., 2008).
Intriguingly, endo-siRNAs weret only in ovaries, but also in
somatic cells like y headsr-2 cultured cells, which are regarded as
y somaticryonic origin (Czech et al., 2008; Ghildiyal et al.,
2008;
t al., 2008a,b). Nuances on the origin and biogenesis ofNAs in
somatic cells have been reviewed (Kim et al.,ura and Lai, 2008;
Ghildiyal and Zamore, 2009).ngly, many endo-siRNAs from germline
and somaticthe same transposons that are targeted by piRNAs
(e.g.
blood, and others; Chung et al., 2008; Czech et al., 2008;t al.,
2008; Kawamura et al., 2008). When taking intoy uniquely mapping
TE-associated siRNAs or normaliz-ciated siRNA reads to the number
of genomic loci, manymatches still accumulate on piRNA cluster loci
(Chung; Czech et al., 2008). However, the number of endo-ping
topiRNAclusters canbeestimated tobeanorderoflower than piRNAs, and
the distribution of piRNAs ver-mapping to consensus TE sequences
differ signicantly.e, over 90% of the piRNAs corresponding to mdg1
andntisense matches, while only 60% of the endo-siRNAsing to these
elements are antisense matches (Chung ett is unclear if endo-siRNAs
are derived from the samenal loci as piRNAs.
piece of piRNA background
in y and zebrash embryos were the rst to describecomplementary
to repetitive transposable elementsthese were named
repeat-associated siRNAs (rasiR-
in et al., 2001, 2003; Elbashir et al., 2001; Chen et al.,e
small RNAs, as a class, have been reclassied as Piwi-RNAs (piRNAs)
because these rasiRNAs and other smallrify with PIWI-group proteins
from ies and mam-
in et al., 2007a; Malone and Hannon, 2009; Ghildiyale, 2009).
The revolution in characterizing piRNAs hasgeneration deep
sequencing technology (Morozova ethich has revealed the
extraordinary sequence diversityl piRNAs in ies and mammals.
Recently, piRNAs haveeeply sequenced in zebrash, frog, monotremes,
silk-ges, and sea anemone (Grimson et al., 2008;Murchison; Armisen
et al., 2009; Houwing et al., 2007; Kawaoka; Lau et al., 2009a),
but studies in y and mouse havest of our understanding of piRNAs
and their genomic
eracting RNAs can be classied into two main typeshere the piRNAs
originate in the genome (Fig. 3). Classedominantly begin with a 5
uridine (U), and generallyely as clusters to genomic regions with a
pronounced, such that single-stranded precursors ranging fromases
(kb) are thought to give rise to a huge variety
-
N.C. Lau / The International Journal of Biochemistry & Cell
Biology 42 (2010) 13341347 1337
Fig. 3. Classi ic regipiRNAs. Class esis ofrom mRNAs ( ion (3the
ribosome f hat pr
of piRNAs.spermatocygressing thrGirard et al.I piRNA
cludivergent trdomains of2006). Maplarge strandloci,
sinceproductionever, thesebecausema
Class II pthey have atransposonbegins withond form csense
stranbase-pair tosense piRNAone PIWI hare antisendominantly(AGO3)
(Bre
When Athe Slicer athe TE tranporation intranscript mpiRNAs
incowell undersmodel has(Fig. 3), becamplify piR2009). Althoclear
whethclass I overto TEs and cpiRNAs areLau et al., 2
MammaMiwi, Mili,germline, w
Wataiwi2ellsoniaspers Mieralumbtestomidst toAs,
tumeclus008)entlyands expternapprrt ofary, a(Lauco pican
btoriecation of Piwi-interacting RNAs. Multi-kilobase clusters of
piRNAs lying in intergenII piRNAs are thought to be generated by a
ping-pong mechanism, whereas biogengenic piRNA precursors) appear
to preferentially arise from the 3 untranslated regor access to
mRNAs or cryptic promoters may reside upstream of certain 3 UTRs
t
These piRNAs are typically abundant in adult rodenttes and
spermatids when the male germ cells are pro-ough
thepachytenestageofmeiosis (Aravinet al., 2006;, 2006; Lau et al.,
2006). Inmammals, someof these classsters exhibit a bi-directional
conguration that impliesanscription, with a 0.11kb region
separating the twopiRNAs (Aravin et al., 2006; Girard et al., 2006;
Lau et al.,ping of class I piRNAs in D. melanogaster also
revealed-biased clusters of piRNAs described as master
controlmutations near these loci have broad effects on piRNAand
transposon control (Brennecke et al., 2007). How-loci also
contribute to the production of class II piRNAsny transposon relics
have accumulated in these regions.iRNAs generally map to multiple
genomic loci becausehigher propensity to match repetitive elements
like
s. These piRNAs are composed of one form that oftena 5 U and is
antisense to TE messages, while a sec-
ontains adenine (A) at position 10 and is the samed as TE
messages (Fig. 3). The class II piRNA forms caneach other in the
rst 10nt, such that the U of the anti-matches the A of the sense
piRNA. In D. melanogaster,
omolog, Aubergine (Aub), mainly binds piRNAs thatse to TEs,
while piRNAs that are sense to TEs are pre-bound by another
piRNA-binding protein, Argonaute-3nnecke et al., 2007; Gunawardane
et al., 2007).ubpiRNA complexes seek transcripts from active
TEs,
2008;Mili, Mgerm cmatogWhenexpresare gensmall nmouseium
brcontraII piRNmore npiRNAet al., 2
Recin iesovarieare maferenta cohothe
ovmencoamentesteslaboractivity in Aub that is guided by the piRNA
can cleavescript to dene the 5 end of a new piRNA for incor-to
AGO3. The AGO3piRNA complex reciprocates on aade from master
control loci to dene the 5 ends ofrporated intoAub. Additional
processing events not yettood help dene the 3 end of the piRNA.
This circularbeen called the ping-pong amplication mechanismause
two PIWI proteins ping-pong off of each other toNA biogenesis (Kim
et al., 2009; Malone and Hannon,ugh Aub and AGO3 clearly bind class
II piRNAs, it is noter Piwi, the founding member of PIWI proteins,
prefersclass II piRNAs. However, its piRNAs are also antisensean
accumulate abundantly evenwhenAGO3-associatedpoorly expressed or
absent (Gunawardane et al., 2007;009b).ls also contain three PIWI
homologs, and in mice theand Miwi2 proteins are all expressed in
the malehereas oocytes appear to express only Mili (Tam et al.,
piRNAs acrmRNAs, suof the mRNadditional cthat remain
5. Nemato
Most anII types; hoelegans andtheir initialdomains ofupstream
oeach indivitranscriptio
At rst glength andons give rise to both class I (primary) piRNAs
and class II (secondary)f class I piRNAs is unclear. However, a
group of class I piRNAs derived UTRs) of the mRNAs, suggesting PIWI
proteins might compete withomote transcription of an independent
transcript.
nabe et al., 2008). In early stages of spermatogenesis,, and
class II piRNAs are expressed in the primordialthat develop and
divide by mitosis into primary sper-(Aravin et al., 2003, 2007b,
2008; Carmell et al., 2007).matocytes enter meiosis to become
spermatids, theywi as well as a burst of class I piRNAs. Class II
piRNAsly more difcult to detect in mouse, perhaps due to theer of
early germ cells, whereas class I piRNAs in adult
es are so abundant that they can be detected by ethid-e staining
(Aravin et al., 2006; Girard et al., 2006). In
y master control loci, which yield both class I and classhe
clusters of mouse class II piRNAs are often distinct,rous, and
smaller in genomic coverage than the class Iters (Aravin et al.,
2007b, 2008; Kuramochi-Miyagawa., newdistinctions in class I
piRNAshave come into focusmice. In contrast to adult mouse testes,
D. melanogasterress abundant levels of class II piRNAs, of which
manyally deposited into the embryo. Three groups using dif-oaches
to partition class II piRNAs have determined thatclass I piRNAs are
present in the somatic follicle cells ofnd many derive from a
master control locus called a-et al., 2009b; Li et al., 2009;
Malone et al., 2009). TheRNAs and bulk of class I piRNA clusters in
mouse adulte considered intergenic piRNA clusters. The Lau and Lais
have now observed that signicant numbers of class I
oss diverse animals can also derive from many specicch that the
piRNAs preferentially map to the 3 UTRsAs (Robine et al., 2009).
These recent ndings underlieomplexities in this germline gene
regulatory pathwayto be uncovered.
de piRNAs are different: 21U RNAs
imal piRNAs conformto the categories of class I and classwever,
an atypical class of piRNAs were revealed in C.named 21URNAs
because of their uniform21nt length,5U, and their genomic origin as
a cohort from largechromosome IV (Ruby et al., 2006). A consistent
motiff the sequence encoding the 21U RNAs was detected fordual 21U
specimen, but whether this motif serves as anpromoteror aprocessing
signal is currentlyunknown.lance, 21U RNAs might seem like
endo-siRNAs in theirthe apparent derivation from either genomic
strand, a
-
1338 N.C. Lau / The International Journal of Biochemistry &
Cell Biology 42 (2010) 13341347
hallmark for a dsRNA precursor. However, three groups
recentlydemonstrated that 21U RNAs are actually Piwi-interacting
RNAs:mutants lacking functional Piwi-related gene-1 (prg-1) lack
21URNAs (Batista et al., 2008; Das et al., 2008;Wang and Reinke,
2008),the immunand the biog2008; Das eistics of 21UPRG-1 exhibacid
sequenand concenexhibit temet al., 2008;of 21U RNAposons in thet
al., 2008represent a
Are we nPerhaps notabundant sppiRNAs obsNAs becameclass I
piRNMiyagawaeof piRNA-lago-2 is genhas been
cletranscriptioembryonicproportionet al., 2009a straightfoever,
focuseorganelles,yield insigh
6. The ridd
Although(Figs. 1 andpiRNA biogtodes demo(Vagin et aDas et
al.,pled from mmodel for abiogenesispiRNAs can2007). Neveprimary
piRnism? Howprecursor tnuclease doindening tularly
demoKlattenhoff
Interestianimals examodicatioOhara et al.al., 2008). Ilase
called DAlthough aessary to stDmHen1/Pi
phenotypes of the null mutant are modest (Horwich et al.,
2007;Saito et al., 2007).
Many PIWI-associated factors and genes genetically
linkedwithpiRNA biogenesis have been described, but their direct
impact on
funcs enA he
u; anLim auc a2006ogs oA b8), w, Kif1et aintentermicsells hor
m2009Nishindin
someal gal coocheownon aen deand9; Rproteof thatoid009asilual
Tbioga et alar f
ogs ren thrumnadow iNAdingrecisthatalian2006tif dce metecton fas
as tPerhcel
er qus inerasechyteneermoprecipitates (IPs) of PRG-1 contain
these small RNAs,enesis of 21U RNAs is Dicer-independent (Batista
et al.,t al., 2008). Although the length and genomic character-
RNAs are quite divergent from other animal piRNAs,itsmany
similaritieswith PIWI proteins besides aminoce similarity. PRG-1
expression is germline-restrictedtrated in germ cell foci called
P-granules, and mutantsperature-sensitive sterility due to germcell
loss (BatistaDas et al., 2008;WangandReinke, 2008). Curiously,
lossexpression only up-regulates the Tc3 class of trans-e worm
germline (see below, Batista et al., 2008; Das
). Thus, 21U RNAs and their loci from chromosome IVn extreme
conguration of a piRNA class.ear the saturation point for discovery
of small RNAs?, since abundant RNAs masked the discovery of the
lessecies inpast deep sequencingefforts. Inmice, the class I
cured initial detection of class II piRNAs, but class II
piR-apparent in neonatal and young testes libraries, when
As were absent (Aravin et al., 2007b, 2008; Kuramochi-t al.,
2008). The Zamore laboratoryhas suggested a classike RNAs
detectable in D. melanogaster heads whenetically ablated and the
background of endo-siRNAsared away (Ghildiyal et al., 2008).
Finally, a new class ofn start site small RNAs have been uncovered
in mousestem (ES) cells, but these species represent a minisculeof
the sequenced libraries (Seila et al., 2008; Fejes-Toth).
Increasing the depth of library sequencing may berward approach to
future small RNA discovery. How-d library construction from specic
stages or cell types,or biochemical fractions of ribonucleoproteins
will alsot into the diversity of small RNAs.
les of germline small RNA biogenesis
our picture of miRNA/siRNA biogenesis is detailed2; Kim et al.,
2009), we only have a few insights intoenesis (Fig. 3). Studies in
ies, zebrash, and nema-nstrated that piRNA production does not
require Dicerl., 2006; Houwing et al., 2007; Batista et al.,
2008;2008); this suggests that piRNA biogenesis is uncou-iRNA and
siRNA biogenesis in animals. The ping-pongmplication of piRNA
biogenesis explains why piRNAdoes not require Dicer and how the
prevalent 5 U ofbe dened (Brennecke et al., 2007; Gunawardane et
al.,rtheless, several questions remain: How does efcientNA
production occur without the ping-pong mecha-exactly are piRNA 3
ends dened? How are piRNA
ranscripts selected? It has been hypothesized that
themain-containing proteins Zuc and Squmight play a rolehe3 endof
piRNAs; however, this remains to bemolec-nstrated (Pane et al.,
2007;Ghildiyal andZamore, 2009;and Theurkauf, 2008; Li et al.,
2009).ngly, the 3 ends of piRNAs (and endo-siRNAs) in allmined are
processed to contain a 2 hydroxyl (2OMe)n (Houwing et al., 2007;
Kirino and Mourelatos, 2007;, 2007; Vagin et al., 2006; Ruby et
al., 2006; Grimson etn D. melanogaster, this is mediated by a
ribose methy-mHen1/Pimet (Horwich et al., 2007; Saito et al.,
2007).
2OMe modication of plant siRNAs and miRNAs is nec-abilize these
small RNAs (Yu et al., 2005), the role ofmet and the 2OMe
modication is unclear because the
piRNAprotein(the RNand sq2007;Vasa, Zet al.,homolin piRNal.,
200(RecQ1(Kotaja
Thetional iproteogerm csingleet al.,2009;The
foutudor,abnormmaternand bibeenshThomshas beAGO3,al.,
200Tudorizationchromet al., 22009;
VindividpiRNANishidmolecuhomol
Givconundated goFirst, hand piRnon-cobe impregionmammet al.,the
mosequenbeen dscriptiregion2008).of germ
Othclusterpolympre-papachytgiven gtion still needs to be
evaluated. For example, among thecoded by the y genes implicated in
piRNA biogenesislicases vasa, spn-E, and armi; the putative
nucleases zucd nuage components krimp and mael; Klattenhoff et
al.,nd Kai, 2007; Pane et al., 2007; Vagin et al., 2006), onlynd
Squ have been shown to interact with Aub (Megosh; Thomson et al.,
2008; Pane et al., 2007). The mousef vasa and mael have also been
genetically implicatediogenesis (Kuramochi-Miyagawa et al., 2004;
Soper ethile several candidate PIWI-associated factors in mice7b,
eIF3a) await further physiological characterizationl., 2006; Lau et
al., 2006; Unhavaithaya et al., 2008).rest in PIWI proteins has set
off a race to identify addi-acting factors. Recently, several
laboratories performingof PIWI protein complexes from y, frog, and
mouseave all simultaneously identied a protein factor withultiple
Tudor domain(s) (Kirino et al., 2009a,b; Laua; Reuter et al., 2009;
Vagin et al., 2009; Chen et al.,da et al., 2009; Vasileva et al.,
2009; Wang et al., 2009).g member of the Tudor gene family is D.
melanogastermutants of which produce sterile progeny and displayerm
cell development due to improper localization ofmponents (Thomson
and Lasko, 2005). The structure
mical function of the Tudor protein domain itself
hastobindsymmetricallydimethylatedarginines (sDMAs;
nd Lasko, 2005), and the presence of multiple sDMAstermined in
the N-terminal regions of Mili, Miwi, Aub,Piwi (Chen et al., 2009;
Kirino et al., 2009a; Nishida eteuter et al., 2009; Vagin et al.,
2009). The association ofins and PIWI proteins may be important for
the local-e PIWI/piRNA complex to germ plasm in oocytes or thebody
in mammalian sperm (Kirino et al., 2009a,b; Lau
a; Nishida et al., 2009; Reuter et al., 2009; Vagin et al.,eva
et al., 2009;Wang et al., 2009). However, ablation ofudor genes in
ies and mice causes varied reduction ofenesis and PIWI protein
stability (Kirino et al., 2009a,b;l., 2009; Reuter et al., 2009;
Vagin et al., 2009). Thus theunction of D. melanogaster Tudor and
vertebrate Tudoremains to be fully elucidated.e diversity of small
RNA types in animal gonads, severals vex the eld regarding how germ
cells and associ-al cells regulate small RNA expression and
production.s the transcription of germ cell-specic miRNA,
siRNA,precursors regulated? The promoter elements for theseRNA
genes are not dened, and motif prediction cane if the transcription
start site (TSS) is unknown. Theseparates the piRNA domains in
bi-directional adultpiRNA clustersmayharbor promoter elements
(Aravin; Girard et al., 2006; Lau et al., 2006). In contrast
toetected upstream of 21U RNAs, however, no obviousotif shared by
mammalian bi-directional clusters hased. A recent analysis of
histone modications and tran-ctors inmouse ES cells has
implicatedCpG-rich genomiche promoters of specic miRNA genes
(Marson et al.,aps this methodology can also ascertain the
promotersl-specic small RNA genes.estions regarding transcriptional
regulation of piRNAclude: Which promoting factors and which RNAs
mediate piRNA precursor transcription? How areene piRNA clusters
regulated differently from adultpiRNA clusters during germ cell
development? Does acell express all piRNA clusters simultaneously,
or does
-
N.C. Lau / The International Journal of Biochemistry & Cell
Biology 42 (2010) 13341347 1339
it pick one or several piRNA clusters to express, analogous to
howlymphocytes or olfactory neurons pick one immunoglobulin geneor
odorant receptor gene, respectively, for expression? This
lastquestionhas recentlybeenexplored ina single-cell analysisof
smallRNAs fromprole of piferent indivexpressed mexpressionbut were
dgests that pbetween di
Overall,understandvitro culturbiochemicabeen
lackininparticulaline that reHowever, twbeen foundPIWI
proteiderives fromlevels of cla2009b). Comfrom the ovboth Siwi (ito
expressSince bothsystems wigenesis andpiRNAs into
7. The imp
Althouget al., 2008eral principdevelopmeproliferateprenatal
anstem cells (ferentiatingcells providcells to entcritically,
pAfterwardsthe sperm iferentiationdelivery venents suchfor
buildingPGCs. Mostthat materngerm plasmwhich blast
8. The in
Althougbroadly impsmall RNAsing into focgermline hafor germ
ce
in nematodes result in deformed oocytes (Knight and Bass,
2001),whereas dcr-1 mutations specically in the D. melanogaster
femalegermline produce GSCs that fail to self-renew but are able to
dif-ferentiate into gametes (Hateld et al., 2005; Jin and Xie,
2007).
ons it miRe thrParkgh th
celor Lt al.,so seemalntiatoliferreguuse mNAsnt fronockrs ard
fot al.,c prorans7). Inmlinmat8; Ht comay r
n (Hthe
RNAlez a
ougrentbotpro
zygoZdicon ofrmu005)roteiell degg a7). Aave eNAsams veis a,
wher, aight
pmere geiRNANAs2004playlevend aXenopus tropicalis oocytes that
compared the overallRNAcluster expressionbetweendifferent eggs
fromdif-idual females (Lau et al., 2009a). Interestingly, each
eggultiple piRNA clusters simultaneously, and the cluster
proles were similar in eggs derived from one motherifferent from
eggs from a different mother. This sug-iRNA cluster expression
proles can vary signicantlyfferent individual animals.our grasp of
piRNA biogenesis still lags behind ouring of miRNA and siRNA
biogenesis, partly because ined cells expressing miRNAs and siRNAs
have facilitatedl analysis, while culture systems containing piRNAs
hadg. SincepiRNAexpression is largely restricted to gonads,r
tomeiotic germcells inmammals, ndingamitotic celltained true
gonadal characteristics seemed daunting.o insect cell lines derived
from ovaries have recently
to endogenously express abundant levels of piRNAs andns. An
Ovary Somatic Sheet (OSS) cell line that likelyD.melanogasterovary
follicle cells expresses abundant
ss I piRNAs because it only expresses Piwi (Lau et
al.,plementing the OSS cells is the BmN4 cell line derivedaries of
the silkworm, Bombyx mori, which expresses.e., silkworm Piwi) and
BmAGO3; thus this line appearsclass II piRNAs predominantly
(Kawaoka et al., 2009).these cell lines also express miRNAs and
siRNAs, thesell be invaluable for functional dissection of piRNA
bio-determining how cells partition miRNAs, siRNAs, anddifferent
AGO/PIWI proteins.
act of small RNAs in germline development
h animal germline development is complex (Cinalli; Lin, 1997;
Seydoux and Braun, 2006), some gen-les have emerged. In the
earliest stages of embryonicnt, primordial germ cells (PGCs) are
sequestered andin a niche that includes somatic support cells.
Duringd juvenile development, PGCs transition into germlineGSCs),
which proliferate by self-renewing and/or dif-into secondary germ
progenitor cells. Somatic supporte signals andmolecules to germ
cells that commit germer meiotic phases like leptotene, zygotene,
and, mostachytene, when synapsis of sister chromatids occurs., germ
cells further differentiate into haploid gametes:nmales and the
oocytes in females. During terminal dif-, sperm shed their
cytoplasm and streamline into DNActors. In contrast, oocytes bulk
up on maternal compo-as energy-rich molecules and materials that
are utilizedthe embryo and for establishing the next generation
ofanimal oocytes become polarized during growth, suchal components
concentrate in special cytoplasm calledand serve as determinants in
the embryo to specify
omeres become PGCs.
uence of miRNAs and endo-siRNAs
h it is known that individual small regulatory RNAs canact
animal development (Ambros, 2004), the impact ofas a class on germ
cell development is only now com-us. Studies where dicer or ago
genes are mutated in theve demonstrated that miRNAs and siRNAs are
crucialll development. For example, dicer (dcr-1) mutations
Mutatidisruptenanc2005;Althouto germAgo-1(Park ecells althese
fdiffereand prdown-
Moand siRdiffereDicer knumberequireTang emeiotisome tal., 200the
gerof speral., 200are nothese mablatioof bothing mi(Gonza2008).
Althis appadicer inable toinitialnull (MinjectiMZdiceet al.,
2PIWI pgerm cin theal., 200may hby miR
In mmiRNAtheresiRNAsHowevNAs, mdeveloways aendo-sof miRet
al.,siRNAsmRNAdcr-2 an loqs, ago-1, and mei-p26, a regulator of
ago-1, all likelyNA function and exhibit similar defects in GSC
main-ough the loss of GSC self-renewal (Forstemann et al.,et al.,
2007; Neumuller et al., 2008; Yang et al., 2007).e role of
themiRNApathwaywasdeemed tobe intrinsicls because somatic support
cells expressing functionaloqs failed to rescue the maintenance of
mutant GSCs2007; Yang et al., 2007), proliferationof somatic
supportemsdependentonmiRNAs (Jin andXie, 2007). Together,e germline
studies suggest that miRNAs down-regulateion genes in order to
promote germ cell self-renewalation. However, the validation of
genes predicted to belatedbymiRNAs in the germline remains to be
explored.ale and female germ cells equally depend on miRNAsfor
proper development, but for reasons that may bem y germ cells.
Although conditional oocyte-specicout mice are infertile, ovary
morphology and oocytee similar to wild-type mice, suggesting that
Dicer is notr mammalian oocyte growth (Murchison et al.,
2007;2007). However, the mutant oocytes are incapable ofgression
because the spindles are malformed, whileposons are activated
(Murchison et al., 2007; Tang etcontrast to females, male mice with
dicer mutations in
e exhibit reduced PGC proliferation and a dramatic lossocytes
developing into round spermatids (Maatouk etayashi et al., 2008).
Nevertheless, these mutant malespletely sterile and produce a few
motile sperm, butesult from germ cells that escape the conditional
geneayashi et al., 2008). As in ies, the somatic support cellsmale
and female mouse germline require a function-pathway to ensure that
germ cells properly developnd Behringer, 2009; Hong et al., 2008;
Nagaraja et al.,
h the importance of miRNAs in y and mice germ cells, zebrash
buck this trend because sh mutants lackingh the zygotic genome and
in maternal contribution areduce viable germ cells that can
fertilize and form ante (Giraldez et al., 2005). The
maternal-zygotic dicer-er) embryos eventually fail to
gastrulateproperly, but ana double-stranded miR-403b duplex into
the one-celltant embryo rescues embryonic development (Giraldez. A
possible explanation for thismay be that piRNAs andns can
compensate for miRNAs and siRNAs in zebrashevelopment, since they
are likely maternally depositednd do not depend on Dicer for
production (Houwing etlternatively,maternal transcripts in
zebrashgermcellsvolved mechanisms that obviate a need for
regulation(Mishima et al., 2006).mals, it is more difcult to
differentiate the impact ofrsus endo-siRNAs on germ cell
development becausesingle Dicer that produces both miRNAs and
endo-ile all mammalian AGOs bind both miRNAs and siRNAs.Drosha
germline mutant, which should only lack miR-indicate if endo-siRNAs
are sufcient for germ cell
nt. In D. melanogaster, the miRNA and siRNA path-netically
separable because Dcr-2 and Ago-2 produces independently of the
Dcr-1 and Ago-1 production(Forstemann et al., 2007; Lee et al.,
2004; Okamura; Tomari et al., 2007). However, the role that endo-in
y development remains mysteriousendogenous
ls can be altered when endo-siRNAs are depleted, yetgo-2 mutant
ies that likely lack most endo-siRNAs do
-
1340 N.C. Lau / The International Journal of Biochemistry &
Cell Biology 42 (2010) 13341347
not appear overtly different from wild-type ies (Lee et al.,
2004;Okamura et al., 2004). TE-associated piRNAs might
compensatefor TE-associated endo-siRNAs in the germline, and
perhaps theendo-siRNA pathway impacts behaviors and responses to
naturalselective prtions.
The impmore evideduction or sdecreased 2Argonautessterility,
pret al., 2009accumulatiogametogenfail to segrevan Wolfswucts of
thegametesansubset of 22gets. Bearinfungi, whichand Moazeway
mightcontrast toprimarily eet al., 2009be highly nucleotidylsible
degradexhibit mishistonemet
9. A conse
RNAi hanomenon otransgenesably from atransgenesled to the
irepetitive ethousandsmutagenic ctrol is essenandRNAi cogenomic
inThis modelsiRNAs in f2007) and rfrom both set al., 2005trigger
for pcation of Rdnematodesthemodel (Okamura an
Transposince, ultimson replicaRNAi pathwpathway tothe PIWI parst
demonhighly elev
and Kuramochi-Miyagawa, 2009). In contrast, mutations
affectingendo-siRNA production in ies do not reveal observable
defects ingametogenesis (Ghildiyal and Zamore, 2009; Malone and
Hannon,2009; Okamura and Lai, 2008). The exact mechanism for how
PIWI
s an, buse tgradlyticctivictivt al.,additnsponal tsilenatedthe7;
Kili mIAPin Dmetethy
his, 2sm,ges,et a
se reat b
gh aer Detwellingandted tsh onscriic daHowd zilt to eerthto Tion
(enfoat arans pAs a
, butt (Baed thdsRNRNATEs,008pathnctiondd th
ly togast, TARo maomete piessures that are typically absent in
laboratory condi-
act of endo-siRNAs on the C. elegans germline is muchnt, as many
mutations that abrogate endo-siRNA pro-tability cause varying
degrees of sterility. Mutants with6G endo-siRNAs (eri-1, rrf-3, and
a doublemutant of theT22B3.2 and ZK757.3) exhibit
temperature-dependentesumably due to overexpression of target genes
(Han). In addition, mutations that affect 22G endo-siRNAn (cde-1,
csr-1, drh-3, ego-1, and ekl-1) result in broad
esis defects because mitotic and meiotic chromosomesgate
properly (Claycomb et al., 2009; She et al., 2009;inkel et al.,
2009; Gu et al., 2009). The protein prod-
se genes appear to coalesce around chromosomes
indblastomeres,whileCSR-1, theAGOprotein thatbindsaG endo-siRNAs,
directly interacts with chromatin tar-g similarity to
heterochromatic siRNAs in plants andguide AGOs tomodulate chromatin
dynamics (Grewal
d, 2003), components of the CSR-1 endo-siRNA path-also direct
methylation on histone H3. However, inheterochromatin targets in
fungi and plants, CSR-1 isnriched on coding genes (Claycomb et al.,
2009; She). Interestingly, the dosage and turnover of CSR-1
mayne-tuned, because mutants in cde-1, which encodes atransferase
that uridylates 22G endo-siRNAs for pos-ation, have increased
levels of 22G endo-siRNAs and
aligned mitotic chromosomes, perhaps due to ectopichylation (She
et al., 2009; vanWolfswinkel et al., 2009).
rved role of keeping transposons at bay
d been recognized as a mechanism to explain the phe-f
cosuppression, where the silencing of multi-copycauses the
silencing of endogenous genes, presum-berrant dsRNAs arising from
the repetitive array of(Henikoff, 1998; Birchler et al., 2000).
This hypothesisdea that RNAi could also silence endogenous
genomiclements like transposons, which can number in theof copies
per element in animal genomes. Given theapabilities of
transposonmobilization, transposoncon-tial for the long-term tness
of almost all organisms,uldhaveevolvedasanelegantmeans to recognize
thesevaders with small RNAs (Goodier and Kazazian, 2008).was
conrmed by the discovery of transposon-directedungi and plants
(Birchler et al., 2000; Zaratiegui et al.,asiRNAs from ies and sh,
all of which appear to arisetrands of transposons (Aravin et al.,
2001, 2003; Chen). These studies suggested that dsRNA is the
commonrogramming RNAi to target transposons, and identi-RP genes
important for transposon control in plants andand the endo-siRNA
pathway in y further supportedGhildiyal and Zamore, 2009;Malone
andHannon, 2009;d Lai, 2008).
son control may be most critical in animal germ cellsately, germ
cells are the essential vehicles for transpo-tion. However, instead
of relying on the conventionalay, animal germs cells have evolved
the PIWI/piRNAregulate transposons. The function and primacy of
thway in controlling transposons in the germline wasstrated by y
mutants that lack piRNAs, which showated TE transcript levels in
ovaries and testes (Siomi
proteincidatedantisenand deof cataSlicer aSlicer aSaito e
Ining traadditioposonis elevL1 andal., 200and mL1 andalteredof
DNADNA mBourccytoplacell sta(Aravin
TheposonsAlthouand othlinks bcompelishedrestriczebraTE
tragenom2008).ziwi anthough
Nevferasesformathelp toNAs thC. eleg21U
RNmentsmutanproposprimethis dssilenceet al., 2piRNAPIWI fu
BeyrevealepossibmelanoTAHREother tlize telgenerad piRNAs silence
transposon transcripts is not fully elu-t it is presumed that PIWI
proteins use piRNAs that areo transposons to recognize TE
transcripts for cleavageation. This hypothesis is supported by the
conservationresidues between PIWI and AGO proteins necessary forty
(Tolia and Joshua-Tor, 2007), and the observation ofity in vitro by
PIWI/piRNA complexes (Lau et al., 2006;2006).ion to the slicing
mechanism postulated for silenc-sons, work on mouse PIWI genes has
suggested anranscriptional silencing pathway formaintaining
trans-cing. In the testes of mili and miwi2 mutant mice,
thereexpression of retrotransposons like the LINE elementLTR
element IAP (Aravin et al., 2007b, 2008; Carmell
eturamochi-Miyagawa et al., 2008). Interestingly, miwi2utant mice
also display loss of DNA methylation atloci, whereas the proles of
IAP-targeting piRNAs arenmt3L knockout mice, which lack a putative
regulatorhyltransferase activity during de novo establishment
oflation patterns in the fetal male germline (Aravin and008).
Although Mili and Miwi are predominantly in theMiwi2 is localized
in the nucleus in specic fetal germand this localization is
dependent on functional Milil., 2008).sults have suggested that
Mili and Miwi2 control trans-oth the transcriptional and
post-transcriptional levels.biochemical interaction among Mili,
Miwi2, Dnmt3L,NAmethyltransferases has not been shown, the
geneticen class II piRNAs and DNA methylation events are, and might
explain how silencing of TEs can be estab-maintained in somatic
cells when piRNA expression iso the germline. In addition to these
ndings in mouse,ocytes decient in Ziwi and Zili also display
elevatedpts, and it is hypothesized that mobilized TEs causemage
that triggers apoptosis (Houwing et al., 2007,ever, it is unknown
if DNA methylation is affected ini mutants, and DNA methylation
mechanisms are notxist in invertebrates likeD.melanogaster and C.
elegans.eless, plant small RNAs clearly guide DNA methyltrans-E
loci, and both entities are critical for heterochromatinZaratiegui
et al., 2007; Chen, 2009). RdRPs in plants alsorce TE silencing by
converting TE transcripts into dsR-e processed into secondary
siRNAs. In this respect, theiRNA pathway shares similarity with
plants. Very fewppear to target the major Tc1 and Tc3 transposable
ele-secondary siRNAs targeting Tc3 are lost in the prg-1tista et
al., 2008; Das et al., 2008). Thus, it has beenat 21U RNAs might
help signal RdRPs in C. elegans toA production from aberrant TE
transcripts (Fig. 2), andbecomes the source of secondary siRNAs
that ultimatelyperhaps at the chromatin level (Batista et al.,
2008; Das). Whereas TE silencing is a conserved function for theway
in animals, the mechanistic details of piRNA andon may be quite
varied among animal species.simply controlling TE mobility, y
genetics has alsoat TE control can extend to telomere regulation
andspeciation of organisms. In the rst example, D.
er telomeres contain specic retrotransposons (HeT-A,T) and TAS
repeats that likely recombine with eachintain telomere length
(George et al., 2006). To stabi-
re structure, the transposons and TAS repeats appear toRNAs
which direct Piwi and Aub to genetically impose
-
N.C. Lau / The International Journal of Biochemistry & Cell
Biology 42 (2010) 13341347 1341
heterochromatic marks onto the telomeres (Savitsky et al.,
2006;Yin and Lin, 2007; Klenov et al., 2007). The heterochromatic
marksthat are induced by piRNAs then repress telomeric TEs from
mobi-lizing and can silence transgenes inserted into nearby
telomeres.piRNA pathity to packexpressioning of tranSavitsky et
A secondpathway isdrome of gofrom a crosisolated labhas been
dfemales to ra homologying this gen1999). Onlynotion thatthat
endowdrome by qet al., 2008;al., 2004). PNAs and hyKalmykova
In additisis mediateas an evolutfertility. Thbe geneticapiRNA
contintact and ththe studymcan have a pbarrier thatalso
provokprogeny heexpressionmals (Lau etemperatursyndrome (whether
pimental con
10. ClassicpiRNA path
Many geously discovScreens fortied aub, cspn-E (Gillemore
recen2007). Thegrouped asbut most ofmally (Gilleal., 1997; Lalso
affect Rity in the oonucleates gend of the opolarized mdisrupted
(C
and Kai, 2007; Pane et al., 2007; Wilson et al., 1996).
Interestingly,the proteins from these germline genes all
co-localize at the nuage,a perinuclear zone in nurse cells believed
to be an active region ofRNA organization (Lim and Kai, 2007;
Klattenhoff and Theurkauf,
Klocdo
pmeve shreve
f DNhoffiggemicroesisn obckgrohofftantlocal9), smac
s. Indth mmalltion (piww dgh oSCs ffertipiwind dtraste celnd
so007n imh ges lik1 (H
adral to tsilen006logougermrmatregus. piwminayet faene rArav1).
Inlocuanti
y abePalumpn-E20041; Tonismncinutatiand
2005way mutants (piwi, aub, spn-E) appear to lose the abil-age
telomeres into stable heterochromatin, allowingand mobilization of
the telomeric TEs and loss of silenc-sgenes integrated near
telomeres (Josse et al., 2007;al., 2006; Yin and Lin, 2007; Klenov
et al., 2007).interesting manifestation of TE control by the
piRNAhybrid dysgenesis, which describes a genetic syn-nadal atrophy
and sterility in hybrid progeny derived
s of wild-type D. melanogaster males and females fromoratory
strains (Kidwell et al., 1977). This syndromeetermined to result
from an inability of laboratoryepress certain transposons. Some
have postulated that-based mechanism could be the system for
mediat-etic phenotype (Chaboissier et al., 1998; Jensen et
al.,recently have many groups begun to converge on thesiRNAs and
piRNAs may be the critical maternal factorsdaughters with the
ability to resist the dysgenic syn-uenching TEs (Blumenstiel and
Hartl, 2005; BrenneckeChambeyron et al., 2008; Pelisson et al.,
2007; Sarot etublished reviews further detail the link between
piR-brid dysgenesis (Malone and Hannon, 2009; Shpiz and, 2009).on
to the facet of transposon control, hybrid dysgene-d by piRNAs and
the PIWI pathway can also be viewedionary force that drives
speciation through modulatinge Hannon laboratory demonstrated that
y strains canlly identical but epigenetically dissimilar on the
basis ofent (Brennecke et al., 2008). Since the piRNA pathway
isebulkofpiRNAsarepresent in sterile dysgenichybrids,ay imply that
subtle interplays betweenTEs andpiRNAsrofound and rapid impact in
generating a reproductivecould ultimately drive animal speciation.
This study
es the question ofwhether piRNAprolesmight impactalth, given
that individual differences in piRNA clustermay be quite prevalent
even between wild-type ani-t al., 2009a). Finally, parameters like
reduced growthe and increased parent age can suppress the
dysgenicKidwell et al., 1977); thus, it will be interesting to
seeRNA proles or TE activity become altered by environ-ditions.
mutants but new twists: y genes affecting theway
nes affecting germline development in y were previ-ered before
they were known to affect piRNA function.female sterility and/or
disrupted oocyte polarity iden-uff, vasa, zuc, squ (Schupbach and
Wieschaus, 1991);spie and Berg, 1995); mael (Clegg et al., 1997);
andtly armi (Cook et al., 2004) and krimp (Lim and Kai,se genes all
affect piRNA accumulation and can bea class of related mutations
where oocytes still form,these oocytes cannot be fertilized and
develop abnor-spie and Berg, 1995; Klattenhoff et al., 2007; Clegg
etim and Kai, 2007; Chen et al., 2007). These mutationsNA
localization mechanisms that help establish polar-cyte. For
example, the maternal mRNA for oskar, whicherm plasm, is not
properly localized to the posteriorocytes in these mutants,
possibly because the normallyicrotubule network between nurse cells
and oocytes ishenet al., 2007; Clegg et al., 1997; Cook et al.,
2004; Lim
2008;How
develories hafail to pbers oKlattenthen traffecthypothnizatiothe
baKlattenble muOskaral., 200tubulepiRNAact wiwhile sregula
Thewith feAlthouadult Gcan betion ofago-1 aIn conin nurscells
aet al., 2has beethrougproteinproteinPal-Bhintegrarole inet al.,
2an anathe y
Spebut theoocytethe gerdivide(Ste) gcytes (al., 200gene,
amarilydestro2006;armi, s2001,al., 200mechaSte sileloqs mmiRNAet
al.,and Etkin, 2005; Lim et al., 2009).piRNAs t into the observed
phenotypes of germlinent mutations? The Schupbach and Therkauf
laborato-own thatmutants that areunable to accumulatepiRNAsnt TEs
from mobilizing, which results in elevated num-A breaks in the
female germline (Chen et al., 2007;et al., 2007; Pane et al.,
2007). The aberrant DNA breaksr damage signaling checkpoint
pathways, which in turntubule network organization. Genetic support
for thiscomes from the suppression of microtubule disorga-served
when checkpoint factors are also mutated inund of a piRNA pathway
mutant (Chen et al., 2007;et al., 2007; Pane et al., 2007).
However, these dou-
s in checkpoint pathways do not suppress the loss ofization
(Klattenhoff and Theurkauf, 2008; Navarro etuggesting that RNA
transport, perhaps utilizing micro-hinery, may be a direct role for
PIWI proteins andeed, a sea urchin and a frog PIWI homolog both
inter-icrotubules (Rodriguez et al., 2005; Lau et al.,
2009a),RNApathways have been shown to rely onmicrotubuleBrodersen
et al., 2008; Parry et al., 2007).i gene was discovered in separate
screens for mutantsividing germline stem cells (Lin and Spradling,
1997).varies initially develop normally in piwi mutants, theail to
self-renewandonly differentiate into oocytes thatlized but are
incompetent for embryogenesis. This func-draws similarity to the
GSC maintenance functions ofcr-1, but themechanism for this
function is still obscure.to the cytoplasmic and perinuclear
localization of Aubls, Piwi is predominantly nuclear localized in
both germmatic support cells (Cox et al., 1998, 2000; Brennecke;
Saito et al., 2006; Lau et al., 2009b). Additionally, piwiplicated
in regulationofmulti-copy transgene silencingnetic or direct
interactions with chromatin-associatede Polycomb group (PcG)
proteins and heterochromatinP1; Brower-Toland et al., 2007; Grimaud
et al., 2006;et al., 2004; Yin and Lin, 2007). Since PcG proteins
arehe self-renewing capacity ofmouse ES cells due to theircing
differentiation genes at the chromatin level (Boyer), it is
tempting to speculate that piwi might also serves function in the
nucleus of GSCs or other stem cells ofline.ogenesis in ies also
depends on piwi and aub function,latorymechanismsare
somewhatdistinct fromthose inimutants lack spermbecause GSCs fail
to self-renew inl niche (Lin andSpradling, 1997), but aubmutant
spermil tomaturebecauseproteincrystals encodedbyStellateepeats
accumulate and probably poison the spermato-in et al., 2001, 2004;
Schmidt et al., 1999; Stapleton etwild-type y testes, the
Suppressor of Stellate [Su(Ste)]s with homology to Ste repeats,
produces piRNAs pri-sense to Su(Ste), which then presumably direct
Aub torrant Ste transcripts (Nishida et al., 2007; Vagin et al.,bo
et al., 1994). The oocyte nuage component genes
, zuc, and squ also inuence Ste silencing (Aravin et al.,; Pane
et al., 2007; Schmidt et al., 1999; Stapleton etmari et al., 2004),
suggesting that the core AubpiRNAoperates similarly in males and
females even though
g is male-specic (Palumbo et al., 1994). Interestingly,ons also
de-repress Ste silencing in addition to
affectingendo-siRNAproduction (Czech et al., 2008; Forstemann;
Okamura et al., 2008b), suggesting that either endo-
-
1342 N.C. Lau / The International Journal of Biochemistry &
Cell Biology 42 (2010) 13341347
siRNAs contribute to Ste silencing, or loqs might participate in
thefunction of all three major small RNA types in y germ cells.
Two newly characterized D. melanogaster mutants with decitsin
the piRNA pathway have recently been shown to display game-togenic
defIsolation ofthe use ofgene in protent with thamplicatioond
mutanII piRNAs, pducing piRN(Klattenhofevolving vamatin imm42AB
piRNAIn contrastinstead appclusters.
Althoughthe ago3 anmutants ismechanismpiRNAs arein these
muallowing copiRNA clusttion of piRN(Lau et al., 2erator of cltwo
classesare fewer vof AGO3 an
11. PIWI p
Mouse mtherstwavwhich the cofmeiosis, cmatocytes aPIWI homothat
PIWI pthe testes ato the apop(Carmell etal., 2004). Mpersists thrin
germ celldpp (Aravinet al., 2004and Miwi extal stage deprophase
owhile miwiAs themutathrough unnia also proand Lin, 200
Mouse Pspermatogofected. Thunot exactlymelanogaste
are dispensable for female fertility (Carmell et al., 2007; Deng
andLin, 2002; Kuramochi-Miyagawa et al., 2004), although
long-termfertility studies of homozygous female mutants have not
beendescribed. Since female germ cells only appear to express
Mili,
roteioocyalianothealianto mdo
alians upsh pmaleed agerm
ascultermntalgerziw
posto aprm cy, thes futddress, sirodt
ore t
ougI proroteior b
ior loelansim(Kla
tinglin emmbe6). Tskarto se
ontraspossmapHouwr theed ach pan and Etkons apath(Cooalizaly
dmpoLinransects similar to those seen in aub and piwi mutants.the
ago3nullmutant by the Zamore laboratory requiredreverse genetics,
possibly due to the location of theximity to heterochromatin (Li et
al., 2009). Consis-e hypothesis of AGO3 acting in the ping-pong
piRNAn pathway, the bulk of class II piRNAs are lost. A sec-t
called rhino was also found to be decient in classrimarily those
deriving from master control loci pro-As from both genomic strands,
like the 42AB cluster
f et al., 2009). Rhino was determined to be a rapidlyriant of
heterochromatin protein 1 (HP1), and chro-unoprecipitation of Rhino
indicated its presence at thecluster (Klattenhoff et al., 2009;
Vermaak et al., 2005).
to a chromatin silencing role attributed to HP1, Rhinoears
necessary to stimulate expression of piRNAs from
much interesting biology remains to be garnered fromd rhino
mutants, an important insight revealed by thesethe partitioning of
class I and class II piRNA biogenesiss (Klattenhoff et al., 2009;
Li et al., 2009). Since class IIdepleted and class I piRNA
biogenesis remains intacttants, the class I piRNAs surface in small
RNA libraries,ndent classication of the amenco locus as a class
Ier. Independent studies looking at maternal contribu-As (Malone et
al., 2009) or examining a follicle cell line009b) have also conrmed
amenco as a primary gen-ass I piRNAs; however, the functional
partition of theof piRNAs in vertebrates remains unclear, since
thereertebrate piRNA mutants and the vertebrate orthologsd Rhino
are not obvious.
roteins and vertebrate gametogenesis
ale germ cells initially develop synchronously beforeeofmeiosis
begins at 10dayspostpartum(dpp), duringlass II pre-pachytene piRNAs
are made. After the onsetlass I piRNAs become the dominant species
in late sper-nd spermatids. Constitutive gene targeting of the
threelogs in mice, Miwi, Mili, and Miwi2, has demonstratedroteins
and piRNAs are essential for male fertility, sincere diminished in
each knockout mutant due specicallytosis of germ cells and not the
somatic support cellsal., 2007; Deng and Lin, 2002;
Kuramochi-Miyagawa etili is expressed as early as PGC and GSC
formation and
oughout spermatogenesis,whileMiwi2 is only detecteds in the 7
days between 15 days post coitus (dpc) and 3et al., 2008; Carmell
et al., 2007; Kuramochi-Miyagawa, 2008). This stage-specic
regulation of Miwi2, Mili,pression correlates with the order of the
developmen-fects in mutants: early spermatocytes arrest in earlyf
meiosis I in miwi2 and mili homozygous knockouts,mutant germ cells
arrest in the round spermatid stage.ntmice age, arrestedgermcells
undergoapoptosis, and,known causes, earlier spermatocytes and
spermatogo-gressively become depleted (Carmell et al., 2007; Deng2;
Kuramochi-Miyagawa et al., 2004).IWI mutant neonates contain
wild-type numbers ofnia, indicating that GSC proliferation is
initially unaf-s, at the physiological level, mouse piwi genes
doshare the same stem cell maintenance role of D.
r piwi. Also, in contrast to y piwi, mouse piwi genes
AGO pmousemammesis inmammmitted
Howmammdependzebraand feprovidity andalso msex
deronmefemalezili, theweeksundergfew getionallPerhapmay
atebrate(Wittb
12. M
Althfor PIWPIWI
pizationpostertheD.mbeyondanismsInteresresultsthe nual., 200more
oestingresult.
In cto tranpiRNA2006;role fodiscussof whinizatioKloc
anmutatipiRNAanismthe locbe highport co
Themote tns and endo-siRNAs may play a redundant role withintes
(Tam et al., 2008; Watanabe et al., 2008). However,oogenesis is
also fundamentally different from oogen-
r vertebrates and spermatogenesis in general, becauseoogonia
proliferate only during gestation and are com-eiosis as primary
oocytes at birth.piwigenes impact femalegermlinedevelopment
innon-vertebrates, where oogenesis, like in D. melanogaster,
on mitotic proliferation and self-renewal of GSCs? Theiwi
homologs, ziwi and zili, are expressed in both malegonads, but
mutant analyses of these genes has not
clear answer. Null mutations in ziwi and zili cause steril-cell
loss during juvenile development, but this event
inizes zebrash (Houwing et al., 2007, 2008). Zebrashination is
not driven by sex chromosomes but by envi-and internal cues;
however, a hypomorph of zili allowsm cells to develop into sterile
oocytes. Compared toi mutant phenotypes are generally more severe:
by 3fertilization (wpf) ziwi mutant germ cells universallyoptosis,
while zili mutant gonads at 6 wpf still retain aells expressing
Ziwi (Houwing et al., 2007, 2008). Addi-zili hypomorph causing
female sterility is male fertile.
ure analysis with the genetically tractable medaka shs how PIWI
proteins directly affect oogenesis in ver-nce medaka sex
determination is genetically speciedet al., 2002).
o piRNAs than transposon silencing?
h transposon control is the primary biological functionteins and
piRNAs, evidence for additional functions forns and piRNAs are also
emerging, such as mRNA local-road gene expression control. For
example, the speciccalization of oskar mRNA and other maternal
factors inogaster oocytemight be regulated by the PIWI pathwayply
the activation of DNA damage checkpoint mech-ttenhoff and
Theurkauf, 2008; Navarro et al., 2009).y, overexpression of piwi in
D. melanogaster femalesbryos with greater Oskar expression, which
increases
r of pole cells, the y equivalent of PGCs (Megosh ethis suggests
that Piwi may directly interact and recruitmRNA to the posterior
end of the egg. It would be inter-e if overexpression of aub or
ago3 recapitulates this
st to the >60% ofD.melanogaster piRNAs correspondingons (Yin
and Lin, 2007), only 1834% of adult vertebrateto repetitive
elements (Aravin et al., 2006; Girard et al.,ing et al., 2007; Lau
et al., 2006). So, what might be thebulk of class I piRNAs?
Translational control has beens a signicant mechanism for germ cell
gene regulation,rt of the cellular mechanism depends upon RNA
orga-d localization events (Fig. 4; Mendez and Richter, 2001;in,
2005; Kotaja and Sassone-Corsi, 2007). aub and piwiffecting oskar
mRNA localization is one example of theway intersecting with a
translational regulation mech-k et al., 2004; Harris and Macdonald,
2001). In addition,tion of PIWI/piRNA complexes in the y germline
mayynamic, involving processing bodies and dynein trans-nents (Lim
et al., 2009; Navarro et al., 2009).laboratory has proposed that
Miwi and Mili may pro-lation of male germline transcripts. Their
support for
-
N.C. Lau / The International Journal of Biochemistry & Cell
Biology 42 (2010) 13341347 1343
Fig. 4. Mecha elemtargets may omiRNA/AGO ctransport alonor more
broad
this hypothpolysomeswith mRNAof translatioGrivna et aMiwi and
Morganelle siRNA procesCorsi, 2007assess the ePIWI homowith
compo(Lau et al.,capable of tplete meiosPerhaps theattributed
tdrive oocytoocyte matThe prevalegerm cellsand the
chrprocessing
If PIWI ppiRNAs fromthis functiomRNAs is ubulges likesubstrates
lnistic models of gene regulation by animal germline small RNAs.
(A) Transposable
perate at the level of mRNA degradation. (B) Many miRNA targets
and potential piRNAomplexes or piRNA/PIWI complexes have the
potential to direct target RNAs to sequestratg cytoskeletal tracks.
(D) siRNAs and piRNAs may have the capacity to inhibit and possibly
at the genomic level through regulation of chromosome segregation
(E).
esis includes co-sedimentation of Miwi and Mili within density
gradients, the association of Miwi and Milis and proteins that bind
mRNA caps, and reduced ratesn initiation inmili knockout testes
(Deng and Lin, 2002;l., 2006a,b; Unhavaithaya et al., 2008). In
spermatids,ili concentrate in the chromatoid body, a
perinuclearmilar to nuage and germ plasm and thought to be ansing
center (Kotaja et al., 2006; Kotaja and Sassone-). Although more
biochemical studies are needed toffect of Miwi and Mili on
translation, the X. tropicalislog, Xiwi, localizes to germ plasm
and also associatesnents of the translation machinery, including
mRNAs2009a). Additionally, in zebrash, a zili hypomorph isransposon
silencing, yet the oocytes are unable to com-is normally, resulting
in sterility (Houwing et al., 2008).meiotic defect in this zili
hypomorph could also be
o a loss of translation regulation of protein factors thate
maturation, since translation regulation is integral touration in
Xenopus laevis (Mendez and Richter, 2001).nce of translation
regulation mechanisms in animaland the commonalities between nuage,
germ plasm,omatoid body suggest there may be a conserved RNAand
translation regulation role for PIWI proteins.roteins do regulate
germ cell mRNAs, then how might
intergenic clusters or TEs mechanistically modulaten? The
substrate recognition rules between piRNAs andnknowndo piRNAs
recognize targets with imperfectmiRNAs, or do they only act upon
perfectly matchedike siRNAs (Fig. 4)? Two recent papers suggest
another
possiblemeprimarily th2009; Saitotrafc jammsuggests
mdevelopmethat this pibroadly conto select mmode of gethese two
nfurther inve
13. Evolut
RNAi isanimals anpossess AGincluding mestingly, fubased on thas
animal Pkingdom oand manysequencesRNAs requiand Gorovset al.,
2008)mal germ cent silencing by piRNAs and siRNAs and silencing of
certain miRNA
and siRNA targets are also regulated at the level of
translation. (C)ion in particular cellular compartments, possibly
through active RNAly activate transcription at the chromatin level,
either at specic loci,
chanismof gene regulationbyPIWIproteins processinge 3 UTRs of
select mRNAs into piRNAs (Robine et al.,et al., 2009). Both studies
uncovered theD.melanogasterRNAas a precursor of primary
piRNAs,which one study
ay regulate downstream genes that enforce follicle cellnt (Saito
et al., 2009). The other study demonstratedRNA biogenesis pathway
from the 3 UTRs of mRNAs isserved from ies to vertebrates and may
have evolvedany specic mRNAs for piRNA processing as a possiblene
regulation (Robine et al., 2009). The implications ofew pathways on
germ cell development remain to bestigated.
ionary and concluding perspectives
an ancient mode of gene regulation, conserved fromd plants to
archaea and protists. Almost all eukaryotesO and Dicer proteins and
a population of small RNAs,iRNAs and siRNAs (Ghildiyal and Zamore,
2009). Inter-ngi and plants lack piRNAs since their AGO
proteins,eir amino acid sequence, do not t in the same subcladeIWI
proteins (Seto et al., 2007). However, the nebulousf protozoans
possess homologs to the PIWI subclade,protozoan small RNAs also
serve to target repetitivelike transposons, although the genesis of
these smallres a Dicer enzyme (Couvillion et al., 2009;
Mochizukiky, 2004; Shi et al., 2004, 2006; Ullu et al., 2005;
Zhang. Although plant germ cells are quite different from ani-ells,
plants also utilize their own repertoire of specic
-
1344 N.C. Lau / The International Journal of Biochemistry &
Cell Biology 42 (2010) 13341347
small RNAs to regulate germline tissue development (Slotkin et
al.,2009). Despite this common theme, the miRNA and siRNA path-ways
in animals are distinct from the piRNA pathway in termsof the
restricted tissue expression, independence from Dicer
forbiogenesis,this distincand more renearly all euand some p
In bilateexample, thsequence le2000). Thisin essentiaing
interact2009). Neithexhibit theThis may bof endo-siRof miRNA/mto a
muchcompared t
Howevethe level ofmosomes bpresence anless, very litrodent
andThis suggesidentity of tposons thatand germlinmals, the pimaster
con
Arecentpoint of theportion of s(Grimson evectensis anthree
PIWItied basedreads that met al., 2008cells of thessponges
anderivativesorganisms2005; Mulleplanarians,sess somatirise to
planrelate to the
The planhomologs, sneoblasts, athis animalAlthough fupending,
thNAs (mainly(Palakodetismedwi-3 inity of planais not affecare
blockeddownof smlevels, sugg
differentiation (Palakodeti et al., 2008), in contrasts to mouse
EScells, which rely on miRNAs for differentiation (Bernstein et
al.,2003). The fact that planaria and basal animals such as
spongesand sea anemones containpluripotentneoblasts/neoblast-like
cells
er wral fuissueearchay
omsssintent
the emand etpiRNals ietectain mSincest derativs or oondpiR
For emponalit
in sonGSventatin culti
althandhas
RNAinnimay
wled
graic Laions
ns oms sup7298yan L
nces
V. TheV, Leegeno, Gaivel c;442:A, Boan gerA, Hanse inA, Kletural
RBiol 2A, La
small;5:33A, Naded Rents iand possibly in nal regulatory
mechanisms. Perhapstion arose with the PIWI pathway evolving
separatelycently in animal lineages, because siRNAs are found
inkaryotes, while PIWI genes are only known in animalsrotozoans
(Seto et al., 2007; Grimson et al., 2008).rian animals, miRNAs can
be highly conserved. Fore miRNA let-7 is perfectly conserved at the
primaryvel between nematodes and humans (Pasquinelli et al.,may be
because miRNAs regulate hundreds of targetsl gene regulatory
networks via imperfect base pair-ions between the miRNA and target
3 UTRs (Bartel,er individual endo-siRNAs nor individual piRNAs
likelydeep primary sequence conservation seen in miRNAs.e
attributed to the scattered biogenesis of multitudesNAs and piRNAs
from a precursor versus a single pairiRNA* from a miRNA hairpin
precursor, and possiblymore limited regulatory role for piRNAs and
siRNAso miRNAs.r, conservation of entire piRNA clusters
ismaintained atsyntenywithinmammals. Syntenic alignments of
chro-etween mouse, rat, and human illustrate the conservedd
conguration of pachytene piRNA clusters. Neverthe-tle primary
sequence conservation is detected betweenhuman piRNAs (Girard et
al., 2006; Lau et al., 2006).ts that piRNA production is essential,
but the sequencehe piRNAs can evolve rapidly, possibly to combat
trans-also evolvequickly. AlthoughPIWIprotein componentse functions
of piRNAs may be conserved among ani-RNA clusters can vary
signicantly (i.e. D. melanogastertrol loci versus C. elegans 21U
RNA clusters).analysis of twonon-bilateriananimals that areat
abasalevolutionary tree has indicated that the major pro-
mall RNAs in basal animals exhibit qualities of piRNAst al.,
2008). The genomes of the anemone Nematostellad the sponge
Amphimedon queenslandica each encodehomologs, and class II and
class I piRNAs could be iden-upon reads that form ping-pong pairs
and clusters ofap in a strand-biased fashion, respectively
(Grimson
). Whether these piRNAs are restricted to the germe
non-bilaterians remains to be established. However,d anemones
contain not typical germ cells, but ratherof somatic pluripotent
stem cells, which lend to theseextraordinary regenerative
capabilities (Extavour et al.,r, 2006). Interestingly, one class of
bilaterian animals,also exhibit extensive regenerative capacities
and pos-c pluripotent stem cells called neoblasts, which can
givearian germ cells. Small RNA studies of planaria mightbiology of
non-bilaterians.arian Schmidtea mediterranea also contains three
piwimedwi-1, -2, and -3, which are mainly expressed in thend both
piRNAs and miRNAs have been cloned from(Palakodeti et al., 2006,
2008; Friedlnder et al., 2009).ll genomic characterization of
planarian piRNAs is stillese piRNAs are a bit longer than other
animal piR-3032nt), and several piRNAs also target transposons
et al., 2008). RNAi knockdown studies of smedwi-2 anddicate that
these genes affect the regenerative capac-ria after amputation,
such that neoblast proliferationted, but differentiation and tissue
repair by neoblasts(Palakodeti et al., 2008; Reddien et al., 2005).
Knock-
edwi-2 and smedwi-3 also reduces piRNAbut notmiRNAesting that
planarian neoblasts rely upon piRNAs for
togethancestulate t
Rescells, mand Thof accepluriporise toand hu(Conraof themammbeen
din certdata).neoblaregenepiRNA
Beytrol byaging.ical coabnormlationbetweeming echrom
In mthe heagationbiologysmallunderpgained
Ackno
I amson, Erdiscusscitatiowork i(HD05Wing-
Refere
AmbrosAmbros
endoAravin A
A no2006
Aravin Amali
Aravin Adefe
Aravin Aa naCell
Aravin AThe2003
Aravin Astranelemith abundant populations of piRNAs suggests
that annction of the piRNA pathway might have been to
mod-regeneration in simple animals.into the biology of pluripotent
stem cells, like ES
enable a revolution in treating human diseases (Yuon, 2008).
Given the ethical and practical limitationsg embryos, other tissue
types have been explored forstem cells, includingmale gonads. Since
germ cells givembryo andGSCs are totipotent, investigations
onmousemale gonads have also yielded pluripotent stem cellsal.,
2008; Kanatsu-Shinohara et al., 2004). The impactA pathway on stem
cell function in vertebrates ands only now being explored, and
while piRNAs have noted inmouse ES cells, class II piRNAs have been
detectedouse spermatogonial stem cell lines (Lau, unpublishedthe
piRNA pathway plays an integral role in GSC andvelopment, it is
encouraging to speculate that othere processes involving stem cells
might be impacted byther germline small RNAs.stem cells, further
investigation of transposon con-NAs may also impact our
understanding of humanxample, maintaining the stability of genomes
is a crit-ent of cancer prevention and avoiding developmental
ies. Comparing TE control in germ cells versus TE regu-matic
cells might reveal the differences in pluripotencyCs and somatic
stemcells. Perhaps epigenetic program-s during gametogenesis via
small RNAs help to establishonformations necessary for proper
embryogenesis.cellular organisms, germ cells may not be essential
toof the individual, but they are essential to the prop-survival of
the species. In the recent past, germ cell
become evidently indispensable for investigations intofunction.
As future experiments reveal the molecularngs of how PIWI proteins
and piRNAs work, the insightslead to applications useful for human
therapies.
gments
teful to Michael Blower, Julie Claycomb, Andrew Grim-i, Sivanne
Pearl, Christina Post, and Dianne Schwarz forand critical reading
of this manuscript. I apologize foritted due to space and
publication constraints. This
ported by a K99/R00 award from theNICHDand theNIH). I dedicate
thismanuscript to thememoryofmy father,au, whose passion for
science lives on in my studies.
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