High-resolution profiling and discovery of planarian small RNAs.

Abstract

Freshwater planarian flatworms possess uncanny regenerative capacities mediated by abundant and collectively totipotent adult stem cells. Key functions of these cells during regeneration and tissue homeostasis have been shown to depend on PIWI, a molecule required for Piwi-interacting RNA (piRNA) expression in planarians. Nevertheless, the full complement of piRNAs and microRNAs (miRNAs) in this organism has yet to be defined. Here we report on the large-scale cloning and sequencing of small RNAs from the planarian Schmidtea mediterranea, yielding altogether millions of sequenced, unique small RNAs. We show that piRNAs are in part organized in genomic clusters and that they share characteristic features with mammalian and fly piRNAs. We further identify 61 novel miRNA genes and thus double the number of known planarian miRNAs. Sequencing, as well as quantitative PCR of small RNAs, uncovered 10 miRNAs enriched in planarian stem cells. These miRNAs are down-regulated in animals in which stem cells have been abrogated by irradiation, and thus constitute miRNAs likely associated with specific stem-cell functions. Altogether, we present the first comprehensive small RNA analysis in animals belonging to the third animal superphylum, the Lophotrochozoa, and single out a number of miRNAs that may function in regeneration. Several of these miRNAs are deeply conserved in animals.

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High-resolution profiling and discovery of planarian
small RNAs
Marc R. Friedla ¨ndera,1, Catherine Adamidia,1, Ting Hanb, Svetlana Lebedevaa, Thomas A. Isenbargerc, Martin Hirstd,
Marco Marrad, Chad Nusbaume, William L. Leee, James C. Jenkinf, Alejandro Sa ´nchez Alvaradof, John K. Kimb,
and Nikolaus Rajewskya,2
aMax Delbru ¨ck Centrum fu ¨r Molekulare Medizin, Robert-Ro ¨ssle-Strasse 10, D-13125 Berlin-Buch, Germany;bDepartment of Human Genetics, Life Sciences Institute,
University of Michigan, Ann Arbor, MI 48109;cDepartments of Bacteriology and Plant Pathology, University of Wisconsin, 1550 Linden Drive, Madison, WI
53706-1521;dGenome Sciences Centre, British Columbia Cancer Center, 675 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3;eBroad Institute of Massachusetts
Institute of Technology and Harvard University, 320 Charles Street, Cambridge, MA 02141; andfDepartment of Neurobiology and Anatomy, Howard Hughes
Medical Institute, University of Utah School of Medicine, 401 Medical Research Education Building, 20 North 1900 East, Salt Lake City, UT 84132
Communicated by Gary Ruvkun, Massachusetts General Hospital, Boston, MA, May 15, 2009 (received for review March 17, 2009)
Freshwater planarian flatworms possess uncanny regenerative ca-
pacities mediated by abundant and collectively totipotent adult stem
cells. Key functions of these cells during regeneration and tissue
homeostasis have been shown to depend on PIWI, a molecule re-
quired for Piwi-interacting RNA (piRNA) expression in planarians.
Nevertheless, the full complement of piRNAs and microRNAs (miR-
NAs) in this organism has yet to be defined. Here we report on the
large-scale cloning and sequencing of small RNAs from the planarian
Schmidtea mediterranea, yielding altogether millions of sequenced,
unique small RNAs. We show that piRNAs are in part organized in
genomic clusters and that they share characteristic features with
mammalian and fly piRNAs. We further identify 61 novel miRNA
genes and thus double the number of known planarian miRNAs.
Sequencing, as well as quantitative PCR of small RNAs, uncovered 10
miRNAs enriched in planarian stem cells. These miRNAs are down-
regulated in animals in which stem cells have been abrogated by
irradiation,andthusconstitutemiRNAslikelyassociatedwithspecific
stem-cell functions. Altogether, we present the first comprehensive
small RNA analysis in animals belonging to the third animal super-
phylum, the Lophotrochozoa, and single out a number of miRNAs
that may function in regeneration. Several of these miRNAs are
deeply conserved in animals.
microRNAs ? miRNAs ? piRNAs ? regeneration ? stem cells
P
biology (1). Planaria are free-living, triploblastic flatworms of the
phylum Platyhelminthes, which is presently considered to belong to
the superphylum Lophotrochozoa. Model systems for modern
molecular and developmental biology have almost exclusively fo-
cused on the other 2 superphyla, i.e., the Deuterostomes (which
includes vertebrates) and the Ecdysozoa (e.g., Caenorhabditis el-
egans and Drosophila melanogaster). Unlike these model systems,
planarians possess remarkable regeneration abilities. Decapitation,
forexample,resultsinthecompleteregenerationoftheheadwithin
7 days after amputation. Such robust restoration of missing body
parts is mediated by adult stem cells known as neoblasts (2). Of the
thousands of known planarian species, Schmidtea mediterranea is
arguably the species of choice for modern molecular biology and
high-throughput, genome-wide approaches because it is diploid, it
existsinsexualandasexualstrains,anditsgenomehasrecentlybeen
sequenced and annotated (3). The size of its genome is roughly a
third of the human genome, and ?80% of the ?20,000 annotated
planarian genes have orthologs in humans. Moreover, by morphol-
ogyalone,neoblastsandtheirimmediatedivisionprogenycomprise
?25%ofallcellsintheadultanimal(4).Inaddition,RNAiscreens
have identified hundreds of genes specifically linked to planarian
regeneration and stem-cell biology (5). Many of these genes are
conserved in humans, and thus understanding planarian regener-
ation promises to yield important insights into human regeneration
and stem cell biology.
lanarians have become a molecularly tractable model system in
which to study regeneration, tissue homeostasis, and stem-cell
In recent years, small, noncoding RNAs have emerged as essen-
tialplayersinalmostallbiologicalprocesses.Manydifferentanimal
small-RNA species have by now been identified, although the
biological functions of these species remain largely unclear (6, 7).
Important exceptions are microRNAs (miRNAs) and Piwi-
interacting RNAs (piRNAs). miRNAs have been shown to play
important roles in many differentiation processes, including regen-
eration (8), whereas at least one function of piRNAs has been
shown to be in maintaining the integrity of the germ line (6). PIWI
proteins are essential for the biogenesis and function of piRNAs,
and they appear to have undergone an expansion in the planarian
genome. We have identified at least 7 likely planarian PIWI genes,
of which 3 (SMEDWI-1–3) have been in part functionally charac-
terized (9, 10). For example, depletion of SMEDWI-2 has been
shown to generate specific defects in stem-cell-mediated regener-
ation and homeostasis (9). Because neoblasts can give rise to
germ-line cells in planaria, it is perhaps not surprising that at least
SMEDWI-1 and SMEDWI-2 proteins are specifically expressed in
neoblasts, and that depletion of SMEDWI-2 or SMEDWI-3 re-
duces piRNA production and both are required for neoblast
function and regeneration (10).
GiventheimportanceofmiRNAsandpiRNAsforplanarianand
stem-cell biology, it is essential to identify and classify small RNAs
in S. mediterranea. Presently, 63 planarian miRNA genes encoding
for 61 unique, mature miRNAs have been identified (11) and
attempts have been made to describe their expression mainly by in
situ hybridization of primary miRNA transcripts (12). However,
maturemiRNAexpressioncanbehighlyregulated(13).Therefore,
to determine the definitive spatial distribution of miRNAs, expres-
sion patterns of primary transcripts have to be complemented by
mature miRNA expression data. Additionally, all known planarian
miRNAs have been identified by classic cloning and Sanger se-
quencing, and it is highly likely that the true number of planarian
miRNAs is much higher. A recent study (10) has further identified
a few thousand piRNAs, which is also almost certainly a vast
underestimate of the true number of planarian piRNAs (14).
We thus used massive, next-generation sequencing methods to
define the full complement of small RNAs present in neoblasts,
Author contributions: M.R.F., C.A., A.S.A., J.K.K., and N.R. designed research; M.R.F., C.A.,
T.H., S.L., M.H., M.M., C.N., W.L.L., J.K.K., and N.R. performed research; M.R.F., C.A., T.H.,
S.L.,T.A.I.,M.H.,M.M.,C.N.,W.L.L.,J.C.J.,A.S.A.,J.K.K.,andN.R.contributednewreagents/
analytic tools; M.R.F., C.A., and N.R. analyzed data; and M.R.F., C.A., and N.R. wrote the
paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Data deposition: The sequences reported in this paper have been deposited in the Gene
ExpressionOmnibus(GEO)database,www,ncbi.nln.nih,gov.geo(accessionno.GSE16159).
1M.R.F. and C.A. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: rajewsky@mdc-berlin.de.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0905222106/DCSupplemental.
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animals depleted of neoblasts, and whole animals. Altogether, we
cloned,sequenced,mapped,andannotatedmillionsofsmallRNAs.
Extensive computational, qPCR, and Northern analyses allowed us
to double the number of known planarian miRNAs, quantify their
expression, and identify a number of mature miRNAs likely to be
involved in stem-cell biology. Furthermore, we were able to study
the expression, genomic organization, and biogenesis features of
planarian piRNAs at a resolution orders of magnitude higher than
any previous studies. Our dataset allowed us to compare planarian
piRNA characteristics with known piRNA features in mammals
and ecdysozoans. Altogether, our work brings the characterization
andannotationofsmallRNAsinplanarianstoadepththatisatpar
with other model systems such as C. elegans.
Results
Comprehensive and Quantitative Deep Sequencing of Planarian Small
RNAs. To profile expression differences of small RNAs, we wished
to compare neoblasts, intact animals, and animals devoid of neo-
blasts with each other. Therefore, RNA was obtained from the
clonal asexual strain CIW4 of S. mediterranea from FACS-purified
neoblasts, intact animals, and irradiated animals in which neoblasts
were eliminated by radiation (1). Each of the 3 samples was
sequenced with 2 different methods. Solexa (Illumina) technology
was used to profile all species of small RNAs (size selection: 18–40
nt). Furthermore, we used the 454 Life Sciences (Roche) technol-
ogytospecificallyprofileDicerproducts(suchasmiRNAs)byusing
a more narrow size selection of 18–25 nt. By using a stringent
mapping procedure (see SI Text), we matched a total of ?4.2
million sequencing reads to ?6.7 million loci in the planarian
genome. Table 1 gives an overview of the 6 deep-sequencing
datasets. We next assessed the samples’ quality by 3 criteria:
coverage, reproducibility, and accuracy of expression quantitation.
Coverage.ToestimatethecoverageofplanariansmallRNAsbythe
sequenced RNAs, we computed the overlap of our mapped reads
with known planarian miRNAs and piRNAs. Previously identified
miRNAs were detected by conventional cloning and sequencing
small RNAs from S. mediterranea whole-body samples with a
median miRNA count of 4 (11). We found all of these miRNAs in
our pooled datasets, with a median of ?9,000 counts (Table S1).
Furthermore, our data contain the lowly expressed ‘‘star’’ miRNAs
for 62 of the 63 miRNA genes. Another recent study reported
?4,800 planarian piRNAs deep-sequenced from whole-body sam-
plesofplanarians(10).Wefound38%ofthesepiRNAsinourdata.
Considering that animal piRNA populations are estimated to
consist of hundreds of thousands of unique sequences (14), it is not
surprising that our sequencing of piRNAs is not fully saturated.
Reproducibility. We compared 2 Solexa datasets obtained by se-
quencingbiologicalreplicateplanariansamples.Foreachplanarian
miRNA in miRBase, we plotted the number of times the miRNA
was sequenced in one sample vs. the other sample (Fig. 1A). The
correlation was almost perfect (Pearson’s correlation ? 0.996),
indicating high reproducibility.
Accuracy of Expression Quantitation. We investigated whether our
deep-sequencing data can accurately quantify differential miRNA
expression. We measured expression fold-changes between intact
and irradiated samples for 35 planarian miRNAs by using our
Solexa data and quantitative PCR in samples from independent
biological replicates (Taqman assay; Methods). We found a strong
correlation between the deep-sequencing data and the qPCR
measurements (Fig. 1B, Pearson’s correlation ? 0.93). We con-
clude that our data are comprehensive, reproducible, and can be
used to quantify miRNA expression across samples.
Planarian Small RNAs Are Predominantly miRNAs and piRNAs. We
next identified the types of small RNAs present in planarians and
quantified their expression in neoblast vs. whole-body extracts. We
hypothesized that the comparison of neoblasts with an untreated
whole-bodysampleshouldidentifysmallRNAspeciesup-regulated
in the planarian adult stem cells. If such species are in fact specific
to neoblasts, we would further expect them to have reduced
expression in the irradiated whole-body sample compared with the
samples from the intact, unirradiated animals. Moreover, these
comparisonswouldallowustodetectartifacts,i.e.,highlyexpressed
small RNAs, that may have arisen as a result of cell dissociation
and/or cell sorting.
Small RNAs in the untreated sample showed a bimodal length
distribution with 2 distinct peaks at nucleotides 22 and 32 (Fig. 2A).
We first selected reads that mapped to known planarian miRNAs
frommiRBaseaswellasournovelmiRNAs(seebelow).Thelength
distributionofthesereadshadasinglepeakatnucleotide22,typical
for miRNAs (Fig. 2B). In fact, these 122 miRNAs (known and
novel) account for the entire 22-nt peak in Fig. 2A, suggesting that
few miRNAs remain to be discovered in S. mediterranea. When
subtracting all reads mapping to annotated miRNAs, rRNAs and
tRNAs, and coding exons, the length distribution forms a distinct
peak at nucleotide 32 (Fig. 2C). We tentatively refer to these
sequences as piRNAs, and will present further evidence for this
classification in the next section on piRNAs. Reads mapping to anno-
tatedcodingexonsdisplayaclearpeakapproximatelyatnucleotide32
(Fig. 2D). However, this is likely an artifact caused by ambiguous
read-mappings and the genome annotation (for discussion see SI Text
and Table S2).
We next estimated the relative abundance of different classes of
small RNAs across the 3 sample types (Fig. 2 E–G, pie charts). The
intergenic piRNA fraction is predominant in sorted neoblasts
(82%), intermediate in the untreated sample (61%), and low in the
irradiated sample (25%). The increased fractions of rRNA and
Table 1. The 6 deep-sequencing datasets derived from untreated
and irradiated planarians and isolated neoblasts
Datasets
Sequencing
platform
Sample
type
Number of
unique mapped
reads
Number of
mapped reads
Number
of loci
1
2
3
4
5
6
All
454
454
454
Neoblast
Untreated
Irradiated
Neoblast
Untreated
Irradiated
25,256
27,461
21,163
86,063
984,459
767,496
1,507,162
63,278
93,412
61,391
91,371
1,784,859
2,050,669
4,144,980
95,951
104,769
85,051
417,646
4,064,381
3,018,709
6,700,894
Solexa
Solexa
Solexa
AllAll
Solexa quantification
of miRNA expression
miRNA read counts, replicate 1
miRNA read counts, replicate 2
Pearson’s
correlation = 0.996
1001000 10000100000 10
100
1000
10000 100000
10
521
0.50.2
10
5
2
1
0.5
0.2
10
Solexa vs. qPCR quantification
miRNA fold change, Solexa
miRNA fold change, qPCR
Pearson’s
correlation = 0.93
AB
Fig. 1.
ducibilityofSolexaquantitationofmiRNAexpression.Eachdatapointrepresents
1miRNA.Samplesareindependentbiologicalreplicatesofirradiatedplanarians.
(B) Solexa vs. qPCR quantitation of miRNA fold-changes. Each data point repre-
sents 1 miRNA. Independent biological replicates were used for the Solexa and
the qPCR quantitation.
Reproducible and quantitative sequencing of small RNAs. (A) Repro-
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short piRNAs in the irradiated sample could be a result of degra-
dation. In contrast, the miRNA fraction is low in the neoblast
sample (4%), intermediate in the untreated sample (30%), and
larger in the irradiated sample (46%).
Comparing the abundance of each class of small RNAs across
different samples requires normalizing contents to a stably ex-
pressed endogenous control. We used miR-71c for library normal-
ization because we observed that this miRNA is robustly and
constantly expressed across our 3 samples based on a quantitative
Taqman assay (SI Text and Fig. 3). By normalizing the total read
counts of miRNAs and piRNAs to the read count of miR-71c, we
were able to estimate the relative expression of small RNAs across
samples (Fig. 2 E–G, bar graphs). Intergenic piRNAs have very
high expression in neoblasts (?10-fold higher than in untreated
whole-body planarians) and low expression in irradiated planarians
(4-foldlowerthaninuntreatedplanarians),consistentwiththeidea
that piRNAs may be up-regulated in neoblasts and their division
progeny, i.e., where PIWI proteins are specifically expressed (10).
In contrast, total miRNA contents appeared roughly constant over
the 3 samples, although the abundances of individual miRNAs
varied. We independently repeated this analysis with 2 other
miRNAs (miR-36 and miR-36c) that appeared roughly constant
and obtained comparable results (see Table S3).
Planarian piRNAs Share Key Features with Mammalian and Fly piRNAs.
To characterize planarian piRNAs, we analyzed deep-sequencing
reads that did not map to annotated miRNAs, rRNAs, tRNAs, or
coding sequences (Fig. 2C). These reads display 2 of the defining
features of mammalian and fly piRNAs (reviewed in ref. 14): a
length distribution peaking approximately at nucleotide 30 and a
diverse population (?1.2 million unique sequences in our Solexa
data). Northern blots validated size and expression of 3 planarian
piRNAs (Fig. 4A).
Planarian piRNAs Display a Clear Tendency to Overlap by 10 Nucleo-
tides. The current model of biogenesis proposes that piRNAs are
generated through iterative PIWI-mediated cleavage of transcripts
with complementary sequence [the ‘‘ping-pong’’ amplification
mechanism(15,16)].Accordingtothismodel,piRNAsthatmapto
opposite genomic strands tend to overlap by 10 nt. We investigated
whether this signature is conserved in planarians. However, this is
difficultbecausemanypiRNAsmaptonumerousgenomicloci.For
instance, if 2 reads map to the same 100 loci, their overlap would
1520 25303540
0
50,000
150,000
250,000
152025303540
0
50,000
150,000
250,000
1520 25303540
0
50,000
150,000
250,000
1520253035 40
0
5,000
10,000
15,000
20,000
Neoblasts (91,371 reads)Irradiated (2,050,669 reads)
Total reads
miRNA reads
piRNA readsreads mapping to
coding exons
Length
Count
Length
Length
Length
Count
CountCount
AB
C
D
E
G
piRNA
expression,
normalized
(>20)
miRNA
expression,
normalized
(0.91)
Untreated (1,784,859 reads)
F
piRNA
expression,
normalized
(2.1)
miRNA
expression,
normalized
(1)
piRNA
expression,
normalized
(0.54)
miRNA
expression,
normalized
(0.91)
exon (5%)
tRNA (<1%)
rRNA (9%)
piRNA
(70%)
miRNA (4%)
piRNA,
short (12%)
exon (5%)
tRNA (1%)
rRNA (3%)
miRNA (30%)
piRNA,
short
(10%)
piRNA (51%)
exon (5%)
tRNA (3%)
rRNA (21%)
miRNA
(46%)
piRNA, short
(14%)
piRNA
(11%)
Fig. 2.
sample.Onlyreadssuccessfullymappedtothegenomeareconsidered.(A)Allreads,(B)miRNAreads,(C)piRNAreads,and(D)readsmappingtocodingexonsareshown.
Note that D is on a different scale. piRNA reads ?25 nt (dark green) have features characteristic for piRNAs (see sections on piRNAs). These features were still present
butweaker(70–80%,seeSIText)fortheremainingpiRNAreads(lightgreen,length17–25nt).(E–G,piecharts)ContentsofRNAspeciesinthethreeSolexadatasets:
(E)neoblasts,(F)untreatedplanarians,and(G)irradiatedplanarians.Darkgreen,piRNAs;lightgreen,shortpiRNAs;darkblue,miRNAs;lightblue,rRNA;yellow,tRNA;
orange,mRNA.(E–G,bargraphs)miRNA(blue),piRNA(darkgreen),andshortpiRNA(lightgreen)expression.The454data,producedbysequencingRNAsthatwere
18–25-ntlong,wereusedtoestimatemiRNAexpression,whereastheSolexadatawereusedforpiRNAexpression.TotalmiRNAandpiRNAreadcountswerenormalized
to miR-71c. miRNA expression of the untreated sample was set to 1, and the other expression bars were scaled accordingly (numbers in parentheses above each bar).
Note that piRNA expression in the neoblast sample is out of scale.
Small RNA contents in planarian neoblasts and whole-body samples. (A–D) Length profiles of Solexa reads from sequencing of the untreated whole-body
.
miRNA expression change
measured by qPCR
log 2 foldchange, irradiated/untreated
let−7a
let−7b
miR-36b
let−7c
let-7d
miR-2160
miR−752
miR−13
miR−71b
miR−2d
miR−2a
miR−71a
miR−2b
miR−2c
miR−71c
bantam−c
bantam−b
bantam−a
miR−756
miR-216
miR-9a
miR-36c
miR−36
miR−92
lin−4
miR-1993
miR-2161
miR-2163
miR-133a
miR−133
miR−1a
miR−1b
miR−1c
miR−10
miR-2151
?3
?2
?1
0
1
Fig. 3.
untreated and irradiated animals was used to quantify expression fold-changes
of 35 miRNAs by qPCR. Data are relative to expression detected for the ubiqui-
tously expressed control ura4 (see Methods). Each miRNA is grouped with its
family members. Horizontal bars indicate miRNA gene clusters.
miRNA expression fold-changes measured by qPCR. Total RNA from
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becounted100times.Thus,toavoidpotentiallyinflatedcounts,we
assigned ‘‘intensities’’ to mappings that were inverse to the number
of mappings for the read. For example, a read mapping to 10 loci
wouldbeassignedanintensityof0.1.Summingoverlapintensitiesover
eachofourdatasets,yieldedmajorpeakswithanoverlapofexactly10
ntintheneoblastandtheuntreatedsample(Fig.4B),whereasthepeak
for the irradiated sample was greatly reduced (Fig. S1).
Planarian Primary piRNAs Map Antisense to Transposons. An impor-
tant function of mammalian and fly piRNAs is to silence trans-
posons. In mouse testes, PIWI proteins cleave transposon mRNA
to generate primary piRNAs, with a uracil in the 5? end (17).
Primary piRNAs base pair with long transcripts that contain
complementary sequence to cleave out secondary piRNAs, which
thus typically have an adenosine at position 10. In fly testes, this is
reversed.PrimarypiRNAsarecleavedfromtranscriptsantisenseto
transposons, and the secondary piRNAs are cleaved from the
transposon mRNA (15, 16).
We found that 32% of the planarian piRNAs map to annotated
transposons (SI Text). piRNAs mapping antisense to transposons
have a clear tendency for a beginning uracil and no other sequence
biases(Fig.4C),indicatingthattheseareprimarypiRNAs.piRNAs
mapping in the sense orientation to transposons have a bias toward
a beginning uracil and an adenosine at position 10.
Planarian piRNAs Locate to Transposons as much as Mouse Pre-
pachytene piRNAs. Mammalian and fly piRNAs differ on the
fraction of the population that is transcribed from transposons.
Mouse pachytene piRNAs have no reported role in transposon
silencing, and map to mouse transposons less than would be
expectedfromthetransposongenomecoverage.Incontrast,mouse
prepachytene piRNAs and fly piRNAs have reported roles in
transposon silencing (reviewed in ref. 18). These map to trans-
posonsasmuch(mouseprepachytene)ormore(fly)thanwouldbe
expected from the transposon genomic coverage.
Planarian transposons cover 31% of the genome and 32% of the
piRNAs. These numbers resemble mouse prepachytene piRNAs.
However, planarian piRNAs do display biases toward particular
classes of transposons. For instance, Mariner elements, active in
planarians (19), have 1.8 times more piRNAs mapping than would
be expected by chance, whereas PiggyBac have half the number of
mapping piRNAs as would be expected (Table S4). These findings
are significant (P ? 0; see SI Text). piRNA transposon association
changes little across the 3 samples.
Planarian piRNA Clusters Display Strand Expression Bias but Seem Not
to Resemble Master Loci. We observed that planarian piRNAs,
similar to those of mammals and flies, tend to map to discrete
regions. To annotate these piRNA clusters, we located 10-kb
regions of the genome to which 100 or more long piRNAs can be
unambiguously traced and where instances of 10-nt overlaps be-
tween such piRNAs occur. This yielded 119 piRNA clusters to
which 6% of all planarian piRNAs in the untreated sample can be
traced (Table S5). These clusters are thus highly (and significantly;
see SI Text) enriched in piRNAs, given that they only constitute
about one thousandth of the planarian genome.
The majority (92%) of planarian piRNA clusters displayed a
strong strand bias, with piRNA mapping intensities 10 times or
higher on one strand (see Fig. S2). piRNAs originating from highly
expressed cluster strands, like primary piRNAs in mouse and fly,
have a strong bias for a 5? uracil, whereas the ones from the lowly
expressed cluster strands, like secondary piRNAs, have a strong
tendency for an adenosine at position 10 (see Fig. 4D). Similar strand
expression biases are observed in the fly ‘‘master loci’’, which are
piRNA clusters densely packed with nonfunctional transposons. How-
ever, we did not identify any master loci in the planarian genome, as
none of the piRNA clusters contained large numbers of transposons.
Discovery and Validation of Novel Planarian miRNAs. To discover
novelmiRNAs,weusedmiRDeep,analgorithmtodetectandscore
Dicer hairpin products such as miRNAs in deep-sequencing data
(20).Varyingscorecut-offsallowtrade-offsbetweensensitivityand
specificity. Sensitivity is computed as the fraction of known miR-
NAs recovered, whereas false positives are estimated by stringent
statistical controls (20).
We separately searched the 454 and Solexa data (SI Text). With
the default cut-off we recovered miRNAs with high sensitivity and
specificity (Fig. 5 A and B). miRDeep identified 70 novel potential
miRNAs, which were further curated (see SI Text). We thus report
a subset of 61 high-confidence miRNAs (Table S6).
We subjected 20 miRNA candidates to Northern blot analysis
and successfully validated 13 of them (see Fig. 5C). Candidates not
observedbyNorthernblottingmaybebelowdetectionthreshold.In
support of this, a more sensitive Taqman assay was used to validate
11 of 11 novel candidates tested (Fig. 3), 4 of which had also been
validated by Northern blot analysis (Fig. 5C). In total, 20 novel
candidates were validated.
The phylogenetic analysis of planarian miRNAs may be partic-
ularly informative as planarians are an outgroup relative to animal
model systems used by the majority of researchers. miRNAs can be
grouped into families based on sequence similarity at their 5? end
(7). Our novel miRNAs increase the number of planarian miRNA
families from 37 to 79 (Fig. S3). The planarian miRNAs share 22
familieswithmammalsand33withfliesandwithnematodes.Thus,
we find planaria to resemble Ecdysozoa (flies, nematodes) more
05 10 1520 2530
0
20000
40000
60000
80000
Overlap in nt
Summed read overlap intensity
All Solexa reads in the untreated sample
A
Solexa reads mapping
anti-sense to transposons
Solexa reads mapping
sense to transposons
Solexa reads from lowly
expressed cluster strands
Solexa reads from highly
expressed cluster strands
1
5
0.0
0.2
0.4
0.6
0.8
1.0
1015
A
C
G
U
BC
1
51015
0.0
0.2
0.4
0.6
0.8
1.0
1
51015
1
510 15
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0D
1
piR:
Northern blot of four piRNAs
20 nts
30 nts
40 nts
4
3
2
Fig. 4.
intensities for Solexa reads in the untreated sample. The horizontal axis is the length of overlap in nucleotides between the 5? ends of reads mapping to the same
genomic locus on opposite strands. The vertical axis shows the intensity of the overlap, summed over the entire dataset. (C and D) Sequence biases of piRNAs. The
horizontal axis shows nucleotide positions from the 5? end, the vertical axis represents nucleotide fractions. (E) piRNAs mapping antisense (Top) and sense (Bottom)
to transposons. (F) piRNAs from highly (Top) and lowly (Bottom) expressed cluster strands.
Features of planarian piRNAs. (A) Northern blot analysis of 4 annotated piRNAs. Bands ?32-nt long are visible for 3 of these piRNAs. (B) Summed overlap
.
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than mammals from the miRNA phylogeny . Interestingly, the
majority (45 of 79) of planarian miRNA families do not show
sequence similarity to known miRNAs. The presence of the miR-
1992family(21)combinedwiththeabsenceofthemiR-1994family
gives evidence to the hypothesis that flatworms are the sister group
to the other lophotrochozoans.
More than a Dozen miRNAs Are Likely Linked to Neoblast Biology. To
identify miRNAs up-regulated in neoblasts, we calculated the
miRNAexpressionfold-changebetweentheuntreatedwholeworm
andisolatedneoblastdeep-sequencingdatasets(SITextandTableS7).
13 miRNAs were up-regulated by ?2-fold in the neoblast sample. As
an independent control, we used qPCR to profile the expression of
these miRNAs in filtered cells enriched in neoblasts vs. untreated
planarians. These were all up-regulated by ?30% in the isolated
neoblasts,whereasanumberofotherprofiledmiRNAsdidnotchange.
To rule out that miRNA up-regulation may have been caused by
cell dissociation or cell sorting, we used qPCR in independently
obtained samples to calculate miRNA expression fold-changes
betweenuntreatedandirradiatedanimals.Wefound10of13ofthe
miRNAs of interest to be ?25% down-regulated in the irradiated
sample (see Table 2 and Fig. 3). These data suggest that a small
subset of miRNAs is significantly up-regulated in neoblasts. Nota-
bly, miRNA genes comprised in clusters (miR-2d, miR-13, miR-
71b,miR-752andlet-7b,miR-36b)aswellasthemiRNAsbelonging
to the same families (let-7, miR-2/miR-13) show a similar differ-
ential expression (Fig. 3).
Interestingly, most of the up-regulated miRNAs in neoblasts
belong to conserved families. The let-7 family has previously been
associated with stem-cell identity. However, previous studies indi-
cate that let-7 is down-regulated posttranscriptionally in stem cells
(22).Inflies,miR-2andmiR-13targettheproapoptoticgenesgrim,
reaper,andsickle(23).Wefindthatall4miRNAsingenomiccluster
containing the planarian miR-2 and miR-13 are up-regulated in
neoblasts,suggestingthatthesemiRNAsareimportantforneoblast
maintenance or neoblast-related function. Additionally, miRNAs
that are typically expressed in specific somatic tissues such as
miR-124 (brain tissues) and miR-1 and miR-133 (muscle tissues)
were down-regulated in neoblasts.
Discussion
By using massive quantitative deep sequencing, we have anno-
tatedsmallRNAspeciesinS.mediterranea.Wehavedoubledthe
number of planarian miRNAs, and have validated a large
fraction of our novel miRNAs. We find that the small RNA-
length peak at nucleotide 22 disappears completely when
miRNAs are removed (Fig. 2), suggesting that a diminishing
number of miRNAs or other Dicer products remain to be
discovered in S. mediterranea. We were also unable to detect
evidence for phased processing of longer transcripts by Dicer.
Moreover, we annotated more than one-million unique piRNA
sequences that locate to genomic clusters. piRNAs have previ-
ously been well-described in Deuterostomes and ecdysozoans
(15, 16, 24–26). We report ?1.2 million unique planarian
20 nts
30 nts
50 nts
40 nts
let-7d
miR-87c
miR-2154miR-754b
miR-87d
miR-124d
?50?25025 50
0
10
20
30
40
50
60
?50?2502550
0
20
40
60
80
100
Known
63
Recovered
52 (83%)
Novel
38
False positives
6 +/- 2
63
53 (84%)
Novel
65
False positives
7+/-3
Known Recovered
miRNAs reported from 454 data by miRDeep
miRNAs reported from Solexa data by miRDeep
miRDeep score
miRDeep score
Counts
Counts
Novel miRNAs reported
>
>
<
<
miR-1992
miR-2156
miR-2151
miR-124e miR-8b miR-36b miR-1993
20 nts
30 nts
50 nts
40 nts
A
B
C
Fig. 5.
miRNAs, dark blue; novel miRNAs, light blue. Known miRNAs below the cut-off are plotted in red (false negatives). The number of false positives was estimated by
miRDeep(20).(B)miRNAsreportedfromSolexadatabymiRDeep;legendsameasA.(C)ValidationofnovelmiRNAs.13miRNAswerevalidatedbyNorthernblotanalysis,
11 by qPCR (Fig. 3). In some cases, miRNA precursors are also detected.
NovelplanarianmiRNAs.(A)miRNAsreportedfrom454databymiRDeep.miRDeepscoresareshownasacolor-encodedhistogram(scorecut-off:1).Known
Table 2. Ten miRNAs up-regulated in neoblasts
miRNAs
Fold change
neo/untr
(454)
Fold change
untr/irr
(qPCR)
Fold change
untr/irr
(Solexa)
let-7a
let-7b, miR-36b
miR-2a
miR-2d, miR-13,
miR-71b,
miR-752
miR-756
miR-2160
2.4
2.1, 3.8
3.7
3.4, 4.9, 3.0, 7.0
3.3
2.5, 2.0
1.4
2.0, 3.3, 2.5, 6.5
5.1
2.1, 0.9
1.0
1.9, 5.5, 2.3, 6.2
2.0
5.0
1.4
2.0
1.6
1.9
miRNAs listed in the same field locate to the same genomic cluster. Neo,
neoblast sample; untr, untreated sample; irr, irradiated sample.
Friedla ¨nder et al. PNAS Early Edition ?
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BIOCHEMISTRY

Page 6

piRNAs locating to more than 100 genomic clusters, and thus
give a first comprehensive description of piRNAs in lophotro-
chozoans.WefindthatpiRNAfeaturescharacteristicforpiRNA
biogenesis (sequence biases, 10-nt overlap) are shared in all 3
metazoan superphylae. Planarian piRNAs also share specific
characteristics with either mammalian or fly piRNAs. Planarian
primary piRNAs, like those in the fly, tend to map antisense to
transposable elements, suggesting that planaria may defend their
genome against transposons similarly to flies. However, from a
different point of view, planarian piRNA biology resembles that
of the mouse more than the fly. First, flatworm piRNAs
associate with transposons as much as mouse prepachytene
piRNAs. Second, the expression of planarian piRNAs is dis-
persed between numerous clusters, similar to what has recently
been observed in the mouse (17). Third, we find no planarian
clusters containing many transposon fragments akin to the
characteristicflymasterloci.Together,ourdataindicatethatthe
piRNA pathway has undergone complex evolution.
We find that at least 10 miRNAs are up-regulated in neoblast
samples. Deep sequencing and qPCR controls show that these are
down-regulated in the irradiated samples depleted of neoblasts,
indicating that they may be specific to neoblast biology. These
miRNAs include all 4 miRNAs from a genomic cluster that
contains miR-2 and miR-13, miRNAs known to inhibit proapo-
ptotic genes in fly (23). We also find that at least 2 members (let-7a
and let-7b) of the highly conserved let-7 family are up-regulated in
neoblasts. Up-regulation of let-7 in neoblast samples was paralleled
by let-7 down-regulation in irradiated samples. These findings are
surprising because let-7 and its family members are known to be
depleted in mammalian stem cells (22, 27) and have been shown in
numerous species to repress cell proliferation and promote differ-
entiation (reviewed in ref. 28). However, recent studies have shown
that cells with the morphological appearance of neoblasts can be
resolved into subtypes, and planarian stem cells may maintain
proliferative activity after commitment (4, 29). Thus, high let-7
levels in neoblast samples may be derived from neoblasts that are
exiting the stem-cell state and committing to a differentiation lineage.
Further let-7 expression analyses, therefore, may help elucidate the
specification of neoblast lineages.
Although planarian stem cells are collectively totipotent be-
causetheycangiverisetoboththesomaticandgermlineagesinthe
adult, our analyses indicate that planarians harbor only 1 miRNA
(miR-92) known to be highly expressed in mammalian embryonic
stem cells (30). However, we found no evidence that its 2 family
membersareup-regulatedinneoblasts.Takentogether,expression
of miRNAs in planarian neoblasts share little if any similarity with
mammalian embryonic stem cells, which may reflect both the adult
nature of planarian stem cells as well as the inherent in vitro versus
invivodifferencesbetweenthese2populationsofanimalstemcells.
Finally, the small RNA profile of neoblasts resemble mouse and
flygerm-linestemcellsinbeingdominatedbypiRNAs.Becausethe
genomic contents of germ-line cells and neoblasts are potentially
immortal, both cell types need to strictly control their genome
integrityduringtransmissiontofuturegenerations,andparticularly,
toprotectitagainsttheuncontrolledpropagationofmobilegenetic
elements. piRNAs have been shown selectively to silence trans-
posons in the fly and mouse genomes (reviewed in ref. 18) and it
islikelythatpiRNAsplaysucharoleinplanaria.Furtherstudiesare
needed to determine whether planarian piRNAs also play a critical
role in epigenetic silencing through DNA/chromatin methylation
like their germ-line homologs (17).
Methods
Sample Preparation and Sequencing. Planarians from the clonal, asexual CIW4
strain of S. mediterranea were starved for 1 week before all experiments. Pla-
narian total RNA was isolated by using TRIzol (Invitrogen). Planarians for the
irradiated samples were exposed to 60 Gy and RNA was extracted 8 days after
irradiation. FACS sorting was performed as described in SI Text. Solexa and 454
sequencing was performed by using the manufacturer’s protocol.
miRNAIdentification.DetectionofnovelmiRNAswasperformedasinref.20.For
more details, see SI Text.
NorthernBlotAnalysisandqPCR.ValidationofmiRNAandpiRNAcandidateswas
performedbyNorthernblotanalysis(TableS8)asdescribedpreviously(31).qPCR
(Taqman miRNA custom assays, ABI) was used to quantify the expression fold-
changeof35miRNAs.cDNAwassynthesizedfrom50ngoftotalRNAfromeither
irradiatedoruntreatedanimals.Sampleswithoutreversetranscriptaseservedas
anegativecontroltemplate.Eachmeasurementwasperformedintriplicate.Two
biological replicates were used. Threshold cycle values are relative to expression
detectedfortheubiquitouslyexpressedcontrolmRNAura4(32).Relativeexpres-
sion of miRNAs is given as log2 of 2???Ctvalues.
ACKNOWLEDGMENTS.WethankAstridFerlinzfromAppliedBiosystemsforthe
custom ABI Taqman primers. Sam Griffith-Jones facilitated the manuscript
through a rapid assignment of miRBase names to the novel planarian miRNAs.
ErikA.SperlingandKevinJ.PetersongavevaluableadviceonmiRNAphylogeny
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