Identification and characterization
of two novel classes of small RNAs
in the mouse germline:
in oocytes and germline small
RNAs in testes
Toshiaki Watanabe,1,2,3,6Atsushi Takeda,4,5Tomoyuki Tsukiyama,1Kazuyuki Mise,4
Tetsuro Okuno,4Hiroyuki Sasaki,2,3Naojiro Minami,1and Hiroshi Imai1
1Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan;2Division of
Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information
and Systems, Mishima 411-8540, Japan;3Department of Genetics, School of Life Science, The Graduate University for
Advanced Studies (SOKENDAI), Mishima 411-8540, Japan;4Laboratory of Plant Pathology, Graduate School of Agriculture,
Kyoto University, Kyoto 606-8502, Japan
Small RNAs ranging in size between 18 and 30 nucleotides (nt) are found in many organisms including yeasts,
plants, and animals. Small RNAs are involved in the regulation of gene expression through translational
repression, mRNA degradation, and chromatin modification. In mammals, microRNAs (miRNAs) are the only
small RNAs that have been well characterized. Here, we have identified two novel classes of small RNAs in
the mouse germline. One class consists of ∼20- to 24-nt small interfering RNAs (siRNAs) from mouse oocytes,
which are derived from retroelements including LINE, SINE, and LTR retrotransposons. Addition of
retrotransposon-derived sequences to the 3? untranslated region (UTR) of a reporter mRNA destabilizes the
mRNA significantly when injected into full-grown oocytes. These results suggest that retrotransposons are
suppressed through the RNAi pathway in mouse oocytes. The other novel class of small RNAs is 26- to 30-nt
germline small RNAs (gsRNAs) from testes. gsRNAs are expressed during spermatogenesis in a
developmentally regulated manner, are mapped to the genome in clusters, and have strong strand bias. These
features are reminiscent of Tetrahymena ∼23- to 24-nt small RNAs and Caenorhabditis elegans X-cluster
small RNAs. A conserved novel small RNA pathway may be present in diverse animals.
[Keywords: Small RNA; siRNA; miRNA; retrotransposon; gsRNA; piwi]
Supplemental material is available at http://www.genesdev.org.
Received March 3, 2006; revised version accepted May 11, 2006.
RNA interference (RNAi) is a sequence-specific gene
regulatory mechanism conserved among diverse eukary-
otes. The sequence specificity in RNAi is determined by
a family of 18- to 30-nucleotide (nt) regulatory small
RNAs (for review, see Aravin and Tuschl 2005). Two
major classes of endogenous small RNAs have been char-
acterized: microRNAs (miRNAs) and small interfering
RNAs (siRNAs). miRNAs—the best-characterized en-
dogenous small RNAs in eukaryotes—have been identi-
fied in diverse plants and animals, and are mainly in-
volved in development and differentiation. miRNAs are
processed from miRNA precursors (pre-miRNAs) with a
through translational repression or mRNA cleavage (for
reviews, see Ambros 2004; Bartel 2004; He and Hannon
2004; Du and Zamore 2005). siRNAs are generated from
long double-stranded RNA (dsRNA) and are mainly in-
volved in defense against molecular parasites including
viruses, transposons, and transgenes through RNAi (Si-
jen and Plasterk 2003; Shi et al. 2004). Endogenous siR-
NAs have been classified into at least three subclasses:
repeat-associated siRNAs (rasiRNAs), trans-acting siR-
NAs (ta-siRNAs), and siRNAs derived from natural an-
tisense transcripts (nat-siRNAs) (Lippman and Martiens-
sen 2004; Peragine et al. 2004; Borsani et al. 2005). ra-
siRNAs corresponding to repetitive elements repress the
repeat sequences at the transcriptional or post-transcrip-
tional level and maintain a centromeric heterochromatic
5Present address: Department of Art and Science, The University of To-
kyo, Tokyo 153-8502, Japan.
E-MAIL email@example.com; FAX 81-55-981-6800.
Article published online ahead of print. Article and publication date are
online at http://www.genesdev.org/cgi/doi/10.1101/gad.1425706.
1732 GENES & DEVELOPMENT 20:1732–1743 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org
structure (Lippman and Martienssen 2004). rasiRNAs
have been cloned and sequenced in Schizosaccharomy-
ces pombe, Trypanosoma brucei, Caenorhabditis el-
egans, Drosophila melanogaster, Danio rerio, and Ara-
bidopsis thaliana, but not in mammals (for review, see
Aravin and Tuschl 2005). In D. melanogaster and
D. rerio, however, the lengths of rasiRNAs are longer
than those of miRNAs (Ambros et al. 2003; Chen et al.
2005). ta-siRNAs are processed from dsRNAs synthe-
sized by an endogenous RNA-dependent RNA polymer-
ase (RdRp) utilizing endogenous mRNAs as template
(Peragine et al. 2004). nat-siRNAs are processed from
dsRNAs formed between endogenous sense and anti-
sense transcripts (Borsani et al. 2005).
In addition to miRNA and siRNA, several classes of
small RNAs have been reported. scanRNAs (scnRNAs;
27–30 nt) in Tetrahymena thermophila are thought to be
derived from dsRNA precursors and guide DNA elimi-
nation (Mochizuki et al. 2002; Lee and Collins 2006).
small RNAs (23–24 nt) in T. thermophila are mapped to
the genome in clusters and are oriented in the same di-
rection (Lee and Collins 2006). X-cluster small RNAs in
C. elegans are derived from a locus on Chromosome X
extending ∼2 kb and are oriented in the same direction
(Ambros et al. 2003). Several factors including DCR-1,
Dicer-related helicase DRH-1, the RdRp RRF-1, and the
exonuclease ERI-1 are reported to be required for the ac-
cumulation of X-cluster small RNAs (Duchaine et al.
2006). The roles of 23- to 24-nt small RNAs in T. ther-
mophila and X-cluster small RNAs in C. elegans remain
Key factors required for the biogenesis and function of
these small RNAs are Dicer and Argonaute proteins
(for review, see Tomari and Zamore 2005). Dicer is an
RNaseIII-like enzyme that recognizes dsRNAs, includ-
ing pre-miRNAs, and processes them into double-
stranded small RNAs (Hutvagner et al. 2001). The Dicer-
generated double-stranded small RNAs are recruited by
Argonaute, and then a strand (called the passenger
strand) is released from Argonaute and the other strand
(called the guide strand) remains associated with Argo-
naute as a guide to regulate gene expression (Matranga
et al. 2005). Based on the amino acid sequence align-
ments, the Argonaute protein family has been subdi-
vided into two subfamilies, referred to as the Argonaute
and Piwi families (Carmell et al. 2002).
Mice have four Argonaute family genes (AGO1–4) and
three Piwi family genes (Miwi, Mili, and Piwil4). AGO1–
4, which are ubiquitously expressed in many tissues (Lu
et al. 2005), recruit miRNAs (Liu et al. 2004). Piwi family
genes are expressed predominantly in male germline
cells (Kuramochi-Miyagawa et al. 2001) and have crucial
roles in spermatogenesis. Disruption of Miwi causes
spermatogenic arrest at the beginning of the round sper-
matid stage (Deng and Lin 2002). Spermatogenesis in
Mili-null mice is blocked completely at the early pro-
phase of the first meiosis (Kuramochi-Miyagawa et al.
2004). The molecular functions and associated small
RNAs of Piwi family proteins remain unknown.
Whether endogenous siRNAs are present in mouse is
unclear for three reasons: (1) There has been no report of
siRNA cloning in mammals; (2) there is no evidence for
the presence of RdRp activity in mammals, which gen-
erates dsRNAs, namely the precursors of siRNAs; and (3)
induction of the interferon pathway by dsRNAs usually
results in cell death, suggesting that mammalian cells
may not tolerate dsRNAs (Elbashir et al. 2001a). How-
ever, in mouse oocytes and preimplantation embryos the
interferon response is suppressed, and injection of long
dsRNAs results in specific reduction in the amount of
target mRNAs (Svoboda et al. 2000; Yan et al. 2005).
Furthermore, retrotransposons and their antisense tran-
scripts are expressed in mouse preimplantation embryos
(Peaston et al. 2004; Svoboda et al. 2004a), and knock-
down of Dicer in mouse preimplantation embryos re-
sults in a 50% increase in IAP and MuERV-L retrotrans-
posons (Svoboda et al. 2004a). These studies suggest that
endogenous dsRNA-induced RNAi can occur in mouse
oocytes and early embryos.
In mammals, hundreds of miRNAs have been identi-
fied by extensive small RNA cloning and bioinformatic
analyses (Lagos-Quintana et al. 2001; Houbaviy et al.
2003; Lim et al. 2003). However, other classes of small
RNAs have not been studied. In this study, as a step to
obtain a whole picture of the small RNA population in
mammals, we have cloned and sequenced small RNAs
from oocytes and testes. We identified two classes of
small RNAs other than miRNAs. One class comprised
which showed characteristics of small RNAs associated
with RNAi. The other class comprised novel 26- to 30-nt
germline small RNAs (gsRNAs) that were present in
male germ cells and had some interesting features that
were distinct from those of siRNAs and miRNAs. The
features of the two classes of small RNAs suggest
their distinct origins and functions and the existence of
two separate small RNA pathways in the mouse germ-
siRNAs in oocytes,
Cloning of small RNAs from the mouse germline
In order to identify small RNAs expressed in the mouse
germline, we constructed small RNA libraries using
RNA isolated from oocytes and testes at different stages.
We collected prophase I full-grown (FG) oocytes and
Metaphase II (MII) oocytes. The seminiferous tubules
from 8-d-old mouse testes contain only spermatogonia
and somatic cells (Bellve et al. 1977). At day 10 after
birth, some spermatogonia start to enter meiotic pro-
phase. Secondary spermatocytes and spermatids appear
at day 18–20. We obtained 8-d-old (spermatogonia and
somatic cells), 15-d-old (from spermatogonia to pachy-
tene spermatocytes and somatic cells), and adult testes
(from spermatogonia to spermatozoa and somatic cells).
We noticed that RNA extracted from the testes of adult
mice contained an abundant population of small RNAs
∼30 nt long that were apparently longer than miRNAs
(Fig. 1). Therefore, we made small RNA libraries by ex-
Novel small RNAs from mouse germline
GENES & DEVELOPMENT1733
cising a portion of a polyacrylamide gel containing ∼16-
to 32-nt small RNAs.
Small RNAs in oocytes
We sequenced a total of 395 clones from oocytes. No
clear difference was observed between the small RNA
profiles of FG and MII oocytes (Table 1). Of these, 247
clones (63%) were mapped to the mouse genome, which
included breakdown products of noncoding RNAs
(rRNAs, tRNAs, snRNAs, snoRNAs, and pseudogene
RNAs; 37.5%), miRNAs (10%), rasiRNAs (11%), and
small RNAs matched to mRNAs (2%). We identified 39
clones of miRNAs derived from seven miRNA genes
(Supplementary Table S1; Supplementary Fig. S1). We
cloned seven small RNAs that matched mRNAs, of
which five were derived from the sense orientation and
two from the antisense orientation. Furthermore, we
cloned two small RNAs that matched the Au76 pseudo-
gene, which was located in the imprinted locus of Igf2r
and antisense to the Air noncoding transcript that is re-
quired for imprinting of Igf2r genes (Lyle et al. 2000;
Sleutels et al. 2002). The unknown small RNAs that did
not match the genome represented 19% in the small
RNA library. This class of small RNAs might be derived
from unidentified bacteria or yeasts, or could result from
We identified 43 clones of rasiRNAs representing 40
different sequences. The 40 unique rasiRNAs were from
11 different retrotransposons (LINE, SINE, and LTR ret-
rotransposons) but not from DNA transposons (see be-
low; Supplementary Table S2). It has been reported that
retrotransposons occupy ∼13% of the expressed sequence
tags (ESTs) in FG oocytes (Peaston et al. 2004). In our
small RNA library, in contrast, rasiRNAs constituted
41% of pol II transcript-derived small RNAs (miRNAs,
rasiRNAs, gsRNAs [see later], other small RNAs derived
from ncRNAs, and mRNAs and unknown small RNAs
that matched the genome). This value was significantly
higher than that of the retrotransposon-derived ESTs
(p < 0.01; Student’s two-tailed t-test), suggesting that ret-
rotransposons are actively processed to small RNAs.
Characteristics of rasiRNAs in oocytes
To investigate whether the rasiRNAs are indeed
siRNAs, we examined their length and first nucleotide.
rasiRNAs in oocytes showed a tight size distribution
between 20 and 23 nt with an average of 21.9 nt (SD =
1.3 nt), similar to that of miRNAs (21.4 ± 1.24 nt; mean
± SD), which are processed by Dicer (Supplementary
Table S3). This size distribution and mean length were
clearly different from those of, for example, the break-
down product of tRNAs (26.4 ± 5.09 nt). In addition, ra-
siRNAs in oocytes showed a strong preference for uri-
dine and adenine residues at the first position (79%)
compared with rRNAs (21%) or tRNAs (45%) (Supple-
mentary Table S4). This preference was consistent with
the trend observed in miRNAs (86%) and functional
siRNAs (Aravin et al. 2003; Khvorova et al. 2003). Fur-
thermore, rasiRNAs in oocytes were mapped to both
sense (24 clones) and antisense (19 clones) orientations of
was enriched by PEG 6000 solution for low-molecular-weight
RNA from various tissues. Approximately 20 micrograms of
low-molecular-weight RNA was loaded on a 15% acrylamide
gel and stained using ethidium bromide. An ∼30-nt RNA band is
observed in testis as indicated by an arrowhead.
Unidentified ∼30-nt small RNA in testis. Total RNA
Number (%) of small cDNAs cloned from mouse oocytes and testes
Stage of oocytes
Stage of testes (days after birth)
SubtotalTotalFGMII 8 d 15 d Adult
Germline small RNA (gsRNA)
Unknown (matched to the genome)
aThis class of small RNAs was mapped to the loci where testis gsRNAs were clustered.
bThis type includes small RNA derived from snRNA, snoRNA, and a pseudogene.
Watanabe et al.
1734 GENES & DEVELOPMENT
retrotransposons (Fig. 2A). These findings suggest that
the rasiRNAs in oocytes are siRNAs processed from
inter- or intramolecular dsRNAs.
Retrotransposon-derived sequences destabilize mRNA
From FG and MII oocytes, we cloned a total of 43
siRNAs, which were derived from L1 (12 clones), MTA
(eight clones), RLTR10 (six clones), IAP1 (five clones),
and others (12 clones) (Supplementary Table S2.). Retro-
transposons are reportedly expressed in a developmen-
tally regulated manner in mouse oocytes and early em-
bryos (Peaston et al. 2004). L1, MTA, and IAP1 were
expressed in FG oocytes, and RLTR10 was selectively
expressed in growing oocytes (Herrera et al. 2005). To
investigate whether these retrotransposons are post-
transcriptionally regulated in FG oocytes, we inserted
L1, MTA, and IAP1 sequences encompassing the siRNAs
(Fig. 2A) into the 3? untranslated region (UTR) of EGFP
mRNA. These chimeric mRNAs were microinjected
into FG oocytes, and the amounts were measured at 16 h
and 40 h after injection. As controls, ∼600 base pairs (bp)
of the 3? UTR of ?-actin mRNA and ∼450 bp of the vector
backbone sequence were respectively inserted into the
same position of EGFP mRNA. All of the EGFP-retro-
transposon mRNAs were degraded significantly more
(P < 0.05; Dunnet) compared with EGFP alone, EGFP-
actin, and EGFP-vector mRNA at 40 h (Fig. 2B). To in-
vestigate whether the instability observed here was
caused by an RNAi mechanism or other mechanisms
such as those involving sequence-specific RNA-binding
proteins (Paillard et al. 1998; Surdej and Jacobs-Lorena
1998), antisense strands of retrotransposons were in-
serted into EGFP mRNA and its degradation was exam-
ined at 16 h after injection. All of these EGFP-retro-
transposons (antisense) underwent more degradation
(P < 0.05; Dunnet) than EGFP mRNA, as was the case for
the sense strands (Fig. 2C). These results are consistent
with the hypothesis that retrotransposons are post-tran-
scriptionally regulated in mouse FG oocytes by an RNAi
Small RNAs in testis
We sequenced a total of 1111 clones from testes at three
stages: 8 d (262 clones), 15 d (450 clones), and adult (399
clones; Table 1). Of the 1111 clones, 994 clones (90%)
matched sequences in the mouse genome, which in-
cluded breakdown products of noncoding RNAs (rRNA,
tRNA, snRNA, snoRNA, and pseudogene; 20%), miR-
NAs (24%), and rasiRNAs (4%). We identified 270 clones
of miRNAs representing 52 kinds of miRNAs, which
included six novel miRNAs. We then confirmed expres-
sion of the six miRNAs by Northern blotting (Supple-
mentary Table S5; Supplementary Fig. S1). Of the novel
miRNAs, four miRNAs (miR-741, miR-742, miR-743,
and miR-465-3p) were mapped to the genome in a 62-kb
region on the X chromosome (Supplementary Fig. S1).
miR-463, miR-465-5p, miR-470, and miR-471, which
were reported to be expressed in testes (Yu et al. 2005),
were also mapped to the same region. In view of the
previous findings on other miRNAs (Lagos-Quintana
et al. 2001; Lau et al. 2001; Houbaviy et al. 2003), some
or all of these eight miRNAs may be transcriptionally
regulated in a polycistronic manner. We identified 46
clones of rasiRNAs representing 44 different sequences
(Supplementary Table S6). The rasiRNAs identified were
from 11 different retrotransposons, but not from DNA
transposons. This specific association with retrotrans-
posons is the same as that of rasiRNAs in mouse oo-
cytes, but the rasiRNAs identified in testes had different
lengths (p < 0.01; t-test) and orientations than those in
oocytes (Supplementary Table S6). This suggests that ra-
siRNAs in testes represent a class of small RNAs that is
and rasiRNAs are mapped. Arrowheads indicate the positions and orientation of the rasiRNAs cloned from an oocyte small RNA
library. The bars under the retrotransposons show the sequences inserted into the EGFP 3? UTR. (B) EGFP, EGFP-retrotransposon,
EGFP-vector, or EGFP-?-actin 3? UTR mRNA was coinjected with DsRed mRNA into mouse FG oocytes. After 0 h, 16 h, and 40 h of
injection, five to 10 oocytes were collected and the injected mRNAs were quantified by RT–PCR. The amount of EGFP was normalized
to that of DsRed and then normalized again to that obtained at 0 h in each case (n = 5; error bars indicate SE). The amounts of
EGFP-retrotransposon mRNAs differed significantly from that of EGFP, EGFP-vector, and EGFP-actin mRNAs at 40 h. (*) P < 0.05
(Dunnet). (C) Insertion of the antisense retrotransposon sequence also reduced mRNA amount (P < 0.05; Dunnet).
Degradation of EGFP mRNA with retrotransposon-derived sequences. (A) Retrotransposons are schematically represented,
Novel small RNAs from mouse germline
GENES & DEVELOPMENT1735
different from the rasiRNAs in oocytes (see the following
A novel class of 26- to 30-nt small RNAs in testes
Most of the remaining sequences from testes were
mapped to the genome in clusters (Fig. 3). Statistically
significant clustering was confirmed at 34 loci (Supple-
mentary Data S1; Supplementary Table S7). In these 34
clusters, a total of 381 small RNAs were mapped, and
each cluster included two to 82 clones (Supplementary
Table S7). These small RNAs were mapped to the ge-
nome in clusters with strand bias (Fig. 3). Most of these
loci were in intergenic regions in the genome and have
not been characterized yet, while some loci overlapped
with known or hypothetical protein-coding genes in the
sense or antisense orientation. Most (86%) of the first
nucleotides of these small RNAs were uridine, as is the
case with miRNAs (76% uridine) (Supplementary Table
S4), but the mapped regions covering these clones were
not folded into the stem-loop structure that is character-
istic of miRNA precursors. It has been reported that one
strand of siRNA duplexes is recruited by AGO, depend-
ing on the asymmetry of internal stability of siRNA
(Khvorova et al. 2003; Schwarz et al. 2003). No fixed
asymmetry pattern was observed for these small RNAs
(data not shown), suggesting that such asymmetry does
not account for the strand bias. The mean length of these
small RNAs (27 ± 2.97 nt) was quite different from that
of miRNAs (21.4 ± 1.24 nt) and rasiRNAs in oocytes
(21.9 ± 1.3 nt) (Supplementary Table S3). The above-
mentioned 381 clones comprised 357 independent se-
quences, and some of these small RNAs overlapped each
other, revealing a high complexity of the small RNA
population (Supplementary Table S7). Their sequences
and genome mapping data did not match those of any
previously described small RNAs. We therefore named
these testes “germline small RNAs” (gsRNAs) (listed in
Supplementary Table S7).
As described in the previous section, a total of 46
rasiRNAs were cloned in testis, the most prominent
classes being IAP (16 clones), B1 (13 clones), and L1 (five
clones) (Supplementary Table S6). The mean length and
first nucleotide of rasiRNAs in testes (25.5 ± 4.53 nt; U:
80%) were similar to those of gsRNAs (Supplementary
Tables S3, S4), but apparently different from those of
gsRNA. The black bars and arrows above or below the genomic DNA denote the locations and orientations of genes and ESTs in the
cluster. Genes are at the top and ESTs are at the bottom. The clusters on AC113269, AC125141, and AC130548 are intergenic regions.
The cluster on AC125187 overlaps a hypothetical protein-coding gene in the same direction. The cluster on AC153373 overlaps two
hypothetical protein-coding genes. Among them, LOC545861 is in the same direction as the cluster, and LOC623016 is in the opposite
direction to the cluster. We did not detect LOC623016 transcripts in testes (Fig. 6C, AC153373, sense primer RT). ESTs with
highlighted accession numbers denote the cDNAs that were examined in Figure 6 by RT–PCR.
Genomic organization of gsRNA clusters. The arrowheads on the genomic DNA denote the orientation and location of each
Watanabe et al.
1736GENES & DEVELOPMENT
rasiRNAs in oocytes (21.9 ± 1.3 nt; U: 51%). Northern
blot analysis using B1 and IAP probes revealed ∼30 nt,
∼26 nt (both IAP and B1), and ∼22 nt (only B1) bands
(Supplementary Fig. S2). Furthermore, an orientation
bias was observed in testes rasiRNAs: All IAP small
RNAs (16 clones) were derived from the antisense
strand, and all B1 small RNAs (13 clones) and all L1
small RNAs (five clones) were from the sense strand
(Supplementary Table S6). These data suggest that the
biogenesis of rasiRNAs in testes is different from that in
oocytes and that gsRNAs and rasiRNAs are generated
through the same pathway in testes.
gsRNA has a 5? phosphate group and a 3? hydroxyl
Upon an electrophoretic separation of RNA from adult
male testes, gsRNAs appeared as a single band at ∼30 nt
(Fig. 1). The RNA from this band was degraded by RNase
treatment but not by DNase, showing that gsRNAs are
indeed RNA (Fig. 4A). T4-RNA ligase catalyzes ligation
of a 5? phosphoryl-terminated RNA to a 3? hydroxyl-
terminated RNA through the formation of a phosphodi-
ester bond. The addition of ligase resulted in the produc-
tion of concatemerized RNA species that migrated
slower than the untreated band (Fig. 4B). The concate-
merized bands were not observed when treated by alka-
line phosphatase (CIAP) before treatment with ligase.
These results indicate that gsRNAs contain a 5? phos-
phate group and a 3? hydroxyl group like other functional
small RNAs (Elbashir et al. 2001b; Mochizuki et al.
gsRNAs are expressed during spermatogenesis
Of nine adult mouse tissues examined, only the testes
expressed the ∼30-nt RNA species (Fig. 1). The testes-
specific expression was confirmed by Northern blotting
using probes complementary to five individual gsRNAs
(Fig. 5A). gsRNAs were detected in testes from a wild-
type mouse, but not in testes from a WV/WVmouse, in
which germ cell differentiation is arrested at spermato-
gonia (Fig. 5B,C; Handel and Eppig 1979), suggesting that
gsRNAs were expressed in more differentiated germ cells.
were gel purified and treated with RNaseA or DNaseI. After the
treatment, RNA was electrophoresed on a denaturing 15%
acrylamide gel and visualized by ethidium-bromide staining. (B)
gsRNAs were incubated with or without CIAP and then treated
with T4 RNA ligase. The right-most lane is untreated gsRNAs.
Molecular characterization of gsRNAs. (A) gsRNAs
weight RNA (20 µg) from various tissues (A), testes of W/WV
and wild-type mice (C), and testes at different stages (E) was
probed for individual gsRNAs. Accession numbers of the clus-
ters are shown on the left in A. gsRNA341 and gsRNA351 are
from the same cluster. Equal loading was confirmed by
ethidium-bromide staining of 5SrRNA and tRNAs. (B,D) Total
RNA (50 µg) from testis of wild-type and WV/WVmutant mice
(B) and wild-type testes at different stages (D) was loaded on a
15% acrylamide gel and stained with ethidium bromide.
gsRNAs appear as a band located at ∼30 nt (arrowhead).
Expression of gsRNAs. (A,C,E) Low-molecular-
Novel small RNAs from mouse germline
GENES & DEVELOPMENT1737
Cloning efficiencies of gsRNAs dramatically changed
among developmental stages: 0% at 8 d, 33% at 15 d, and
59% at an adult stage (Table 1). This indicates that ex-
pression of gsRNAs is developmentally regulated during
spermatogenesis. Indeed, gsRNAs were detected in tes-
tes from 3 wk to adult, but not in testes from 1 and 2 wk
(Fig. 5D). Northern blotting data of five individual
gsRNAs showed the same patterns (Fig. 5E). Expression
of these gsRNAs started at ∼3 wk, peaked at 4 wk, and
declined in adults. In testes of 2- to 3-wk-old mice,
pachytene spermatocytes become abundant. Round sper-
matids become abundant in 3- to 4-wk-old mice, and
adult testes contain all the cell types, including abun-
dant elongated spermatids (Bellve et al. 1977). Thus, the
data indicate that gsRNAs are expressed from the pachy-
tene spermatocyte stage to the round spermatid stage
Interestingly, Northern blotting for gsRNA341 and
gsRNA351 showed a ∼26-nt band in addition to the ∼30-
nt band (Fig. 5E). A faint ∼26-nt band was also observed
in 2-wk-old testes. As for gsRNA351, the ∼26-nt band
was as abundant as the ∼30-nt band at 3 wk, but in adults
the main class was ∼30-nt RNAs. In agreement with the
existence of two size classes of gsRNAs, the gsRNAs
cloned from 15-d-old mice were shorter (26.0 nt) and
more broadly distributed (SD = 3.41 nt) than the gsRNAs
cloned from adults (27.7 ± 2.43 nt) (Supplementary Table
S3). In addition, the size of the most abundantly cloned
class was 26 nt in 15-d-old mice and 30 nt in adults,
suggesting that the gsRNA biogenesis pathway is com-
Primary transcripts of gsRNAs
Some ESTs were mapped to the loci where gsRNAs were
clustered. Interestingly, most of these transcripts were
cloned from testes and were oriented in the same direc-
tion as gsRNAs (Fig. 3). RT–PCR analysis of five of
these transcripts also revealed testis-specific expression
(Fig. 6A; for transcripts examined, see Fig. 3). Further-
more, their expression in testes preceded that of gsRNA
by 1 wk and changed with development in the same
manner as gsRNAs (Fig. 5D). Thus, the ESTs were first
detected at 2 wk, peaked at 3 wk, and declined in adults
tissues (A) and in testes at different stages (B). Accession numbers of the gsRNA clusters are listed on the left. The examined ESTs are
indicated in Figure 3. (C) Strand-specific expression of putative gsRNA precursors. Total RNA from adult mouse testes was reverse-
transcribed with no primer, with a gene-specific sense primer, or with a gene-specific antisense primer, and then PCR was performed
using a set of gene-specific primers. Accession numbers of the gsRNA clusters and the examined ESTs are as in A and B. Retrotrans-
posons (IAP1 and LINE1) and protein-coding genes (?-actin and Histone H2 afx) serve as a positive and a negative control for the
existence of antisense transcripts, respectively. (D) Overrepresented motifs in the putative gsRNA precursors are shown. The motif
discovery tool MEME was used to search for consensus motifs in all 18 putative gsRNA precursor sequences. The numbers of the
gsRNA precursors that contained the consensus motif and E-values are also shown.
Analysis of putative gsRNA precursors. (A,B) Expression of putative gsRNA precursors was analyzed by RT–PCR in various
Watanabe et al.
1738 GENES & DEVELOPMENT
(Fig. 6B). Collectively, these data suggest that the EST
transcripts that we examined are the precursor mol-
ecules of the gsRNAs.
The extreme strand bias in the production of gsRNAs
unlinked to stem-loop structure suggests that the bio-
genesis pathway of gsRNAs is distinct from that of
miRNAs and siRNAs. In support of this suggestion, we
did not detect antisense transcripts of these five tran-
scripts by RT–PCR in 3-wk-old and adult testes (Fig. 6C;
data not shown). To see if gsRNA precursors contain
common secondary structures or motifs, we first
BLASTed all of the gsRNAs against the EST databases
and obtained putative gsRNA precursor sequences. Most
of these sequences had been cloned from testes, but
some were from brain or other tissues. Of these, we ana-
lyzed the sequences of all 18 cDNA clones that were
obtained from testes (Supplementary Table S8). The sec-
ondary structures of the putative precursor sequences, as
predicted by Mfold version 3.2 (Zuker 2003), did not con-
tain stem-loop structures, intramolecular dsRNA, or
other common secondary structures around the gsRNAs.
The motif discovery tool MEME (Bailey and Elkan 1994)
found a consensus sequence, a 15-nt stretch of adenosine
residues (including two guanosine residues), in the inter-
nal regions of 10 sequences (Fig. 6D). Some putative pre-
cursor sequences had longer poly(A) tracts. A search of
18 randomly selected mRNAs failed to find the poly(A)
motif (data not shown). Usually, SINE and LINE retro-
transposons contain a 3? poly(A) tract in their genomic
sequences, which is characteristic of retrotransposal in-
tegration (Maestre et al. 1995). To investigate whether
putative gsRNA precursors contain retrotransposon se-
quences, we used the Repeat Masker program. Of the 18
precursor sequences that we examined, SINE sequences
were observed in six precursors, LINE sequences in five
precursors, and LTR retrotransposon sequences in four
precursors (Supplementary Table S8). A search of 18 ran-
domly selected full-length EST sequences failed to find
(mean = 1, p < 0.01; t-test). Partial retrotransposon se-
quences were abundantly observed in putative gsRNA
In mammals, miRNAs are the only small RNAs that
have been studied in detail. In this study, we identified
two classes of small RNAs in the mouse germline
through cloning and sequencing of the small RNAs of a
broad size distribution. One of these classes is siRNAs in
oocytes and the other is gsRNAs in testes. The unique
features of the two classes of small RNAs reveal that the
pathways and functions of small RNAs in mouse are
more diverse than was previously known.
Regulation of retrotransposons through RNAi
in mouse oocytes
Little is known about endogenous siRNAs in mammals,
even though they have been extensively studied in other
animals and plants. Mammalian cells are not thought to
have endogenous siRNAs, which are processed from
dsRNAs, because dsRNAs >30 nt in length induce the
interferon pathway, leading to cell death (Elbashir et al.
2001a). However, we suspected that functional endog-
enous siRNAs are present in mammalian oocytes be-
cause mammalian oocytes do not have an interferon re-
sponse, and also because RNAi induced by exogenous
long dsRNAs has been observed in mouse oocytes (Svo-
boda et al. 2000; Yan et al. 2005). As expected, we iden-
tified rasiRNAs in mouse oocytes that are likely to be
involved in RNAi. They had a bias for uridine at the first
nucleotide and a narrow size distribution centered at 22
nt. The identification of rasiRNAs and the instability of
chimeric mRNAs containing retrotransposon-derived se-
quences (Fig. 2) suggest that, in mouse oocytes, retro-
transposons are suppressed through RNAi at the post-
transcriptional level. In support of this view, an increase
of retrotransposon transcripts has been reported in
Dicer-deficient embryonic stem (ES) cells and Dicer-
knockdown preimplantation embryos in mouse (Svo-
boda et al. 2004a; Kanellopoulou et al. 2005). In addition,
rasiRNAs in oocytes may regulate the expression of
mRNAs containing retrotransposon-derived sequences.
rasiRNAs have a broad range of mRNAs as putative tar-
gets. This is because retrotransposons act as the first
exon for some genes in oocytes (Peaston et al. 2004), and
18.4% of the mouse genes contain transposable elements
in their untranslated regions (van de Lagemaat et al.
The RNAi pathway causes epigenetic modifications,
such as DNA methylation and histone modification, at
loci homologous to the small RNAs in several organisms
(Volpe et al. 2002; Zilberman et al. 2003; Grishok et al.
2005). Although siRNA-mediated DNA modification
and chromatin remodeling are controversial in mam-
mals (Morris et al. 2004; Park et al. 2004; Svoboda et al.
2004b; Kanellopoulou et al. 2005; Murchison et al. 2005),
it is tempting to speculate that differential expression of
retrotransposons and subsequent production of siRNAs
trigger sequential chromatin remodeling through an
RNAi pathway in mouse oocytes and early embryos.
Pathway and function of gsRNAs in testis
We found a novel class of small RNAs named gsRNAs in
mouse testes. gsRNAs are 26- to 30-nt small RNA mol-
ecules having features clearly different from those of
miRNAs and siRNAs: gsRNAs are longer than miRNAs
and siRNAs and have a strand bias unlinked to a stem-
loop structure. These features suggest that the biogen-
esis pathway of gsRNAs is different from that of
miRNAs and siRNAs. In support of this view, we did not
detect the antisense transcripts of gsRNA precursors by
RT–PCR or antisense small RNAs of gsRNAs by North-
ern blotting (Fig. 6C; data not shown). However, we can-
not exclude the possibility that gsRNAs are of dsRNA
origin, because such precursors may be unstable and
therefore cannot be detected. In fact, X-cluster small
RNAs in C. elegans, which also show strong strand bias,
Novel small RNAs from mouse germline
GENES & DEVELOPMENT1739
may be of dsRNA origin because their biogenesis re-
quires both DCR-1 and RdRp (Duchaine et al. 2006). On
the other hand, we frequently observed retrotransposons
in gsRNA precursors. Although the gsRNA biogenesis
pathway is unclear, one possibility is that mRNAs that
contain retrotransposon-derived sequences in testis are
digested by an unidentified enzyme into 26- to 30-nt
gsRNAs. Alternatively, gsRNAs may originate from
double-stranded RNA–DNA complexes that are pro-
duced by the RT enzymes of retrotransposons. Interest-
ingly, the DNA strand of small RNA–DNA complexes is
transferred to an archaeal Piwi protein (Ma et al. 2005;
Yuan et al. 2005). Piwi family proteins (Miwi, Mili,
Piwil4), which are expressed in testis and are required for
spermatogenesis, could be involved in the gsRNA path-
Expression of gsRNAs was found to be limited to germ
cells in testis and limited to the period from the pachy-
tene spermatocyte stage to the early spermatid stage.
Northern blotting with the gsRNA341 and gsRNA351
probes showed the existence of ∼30-nt long gsRNAs and
∼26-nt short gsRNAs (Fig. 5E). Interestingly, the short
gsRNAs were faintly detected at 2 wk, when the long
gsRNAs were not yet detected. These results suggest
that gsRNAs occur in two distinct classes and that short
gsRNAs are expressed earlier than the long gsRNAs. In
support of the existence of both long and short gsRNAs,
the sizes of the gsRNAs obtained from 15-d-old testes are
shorter than those obtained from adult testes (Supple-
mentary Table S3). The existence of two classes of
gsRNAs suggests that two distinct biogenesis pathways
with different gsRNA-generating enzymes and gsRNA-
binding proteins are present in mouse testis. gsRNAs,
which are probably derived from single-stranded RNAs,
are unlikely to be transferred to AGO1–4, which are
thought to recognize only small RNA duplexes. Interest-
ingly, in mouse testes, the expression patterns of long
gsRNAs are very similar to those of Miwi, and the ex-
pression of short gsRNAs overlaps with Mili (Kuramo-
chi-Miyagawa et al. 2001). Examination of the associa-
tion of gsRNAs with Piwi proteins may shed light on the
biological roles of gsRNAs in mouse testis.
Most gsRNAs are not complementary to protein-cod-
ing mRNAs and not conserved among species. There-
fore, they are unlikely to regulate mRNAs in a post-
transcriptional manner as miRNAs do. It is important in
the future to identify the proteins associated with
gsRNAs and to determine the biological processes in
which gsRNAs are involved.
Materials and methods
Small RNA preparation and cDNA library construction
Total RNA was isolated from C57/B6 mouse testes or oocytes
using the guanidium thiocyanate-phenol-chloroform method.
Testes were isolated from 8-d-old, 15-d-old, and adult male
mice. Females were injected with eCG (5 IU) 48 h before oocyte
collection, and then FG oocytes were collected from ovaries as
described previously (Viveiros et al. 2004). For MII oocytes, fe-
males were superovulated by injection of eCG (5 IU) followed by
an injection of hCG (5 IU) 48 h later, and then oocytes were
collected the next morning and incubated in PBS containing 3
mg/mL PVP and 0.3 mg/mL hyaluronidase to remove follicle
cells (Nagy et al. 2003). To isolate small RNAs, 50 µg of total
RNA from testes and ∼1.5 µg of total RNA (∼1200 oocytes) from
oocytes was used. Small RNAs were cloned using the basic
protocol (Pfeffer et al. 2005).
To classify small RNAs, we performed NCBI BLASTn searches
(http://www.ncbi.nlm.nih.gov) using the default settings. Com-
plexity filtering was used in these BLAST searches, and we
chose the “nr” database from all organisms. A small RNA se-
quence was considered to match the genome if it had no more
than two mismatches or if it had a 16-nt stretch of exact match.
Most of the sequences were classified in this search. The re-
maining sequences that were matched to sequences in the
mouse genome were then further classified using the following
tools or databases: miRNA Registry (http://microrna.sanger
.ac.uk/sequences/index.shtml) and Mfold 3.2 (http://www
.bioinfo.rpi.edu/applications/mfold) for miRNAs; tRNAscan-SE
(http://www.genetics.wustl.edu/eddy/tRNAscan-SE) for tRNAs;
and Repeat Masker (http://www.repeatmasker.org) and L1Base
(http://l1base.molgen.mpg.de) for repeat sequences. The small
RNAs that were mapped to the mouse genome in clusters and
not classified as noncoding RNAs or retrotransposons were clas-
sified as gsRNAs. In cases where a small RNA was classified as
both gsRNA and mRNA, the small RNA was classified as
To identify gsRNA precursor sequences, all gsRNAs were
BLASTed against the “nr” and “est” databases from all organ-
isms, and then the EST sequences cloned from mouse testes
were used for further analysis. RNA folding was predicted with
Mfold 3.2. After removal of the poly(A) tail, consensus motifs
were analyzed by MEME (http://meme.nbcr.net/meme/intro
.html) using the “any number of repetition” model. Repeat se-
quences were analyzed using the Repeat Masker program.
A cDNA fragment of EGFP was amplified by PCR from plasmid
pd2EGFP-1 (Clontech) using the primers pd2EGFP sense and
pd2EGFP antisense (Supplementary Table S9). The amplified
fragment was digested with SalI and EcoRI, and ligated to the
SalI and EcoRI site of pBlueScript SK−(Stratagene) to construct
pBS-EGFP. To insert the retrotransposon-derived sequences or
3? UTR of ?-actin downstream of the EGFP sequence in pBS-
EGFP, retrotransposon-derived fragments and the 3? UTR of
?-actin were amplified using cDNA that was reverse transcribed
from mouse MII oocyte RNA using an oligo(dT) primer. The
primers used in RT–PCR are listed in Supplementary Table S9.
The retrotransposon-derived fragments and 3? UTR of ?-actin
were cloned into the pCR4 Blunt-TOPO vector (Invitrogen), and
then digested with EcoRI and ligated to the EcoRI site of pBS-
EGFP. For DsRed mRNA, pDsRed1-N1 (Clontech) was digested
with SalI and NotI, and then ligated to the SalI and NotI sites of
pBlueScript SK−. The constructed plasmids were digested with
NotI, and then mRNAs for EGFP, EGFP-actin, EGFP-IAP1
(sense and antisense), EGFP-MTA (sense and antisense), EGFP-
L1 (sense and antisense), and DsRed were synthesized using the
mMessage mMachine T7 ultra kit (Ambion) according to the
mRNA, pBS-EGFP was digested with AflIII.
FG oocytes from B6D2F1, mice were injected with 5–10 pL
of either EGFP, EGFP-actin, EGFP-vector, EGFP-IAP1, EGFP-
Watanabe et al.
1740GENES & DEVELOPMENT
MTA, or EGFP-L1 mRNA solutions (50 nM) containing DsRed
mRNA (2.5 µM) as described (Nagy et al. 2003). The RNA-in-
jected oocytes were cultured in CZB medium at 37°C under 5%
CO2in air. At 0, 16, and 40 h post-injection, five to 10 oocytes
were collected, and then RNA was extracted and reverse tran-
scribed using oligo(dT) primers. For quantitative analysis, EGFP
and DsRed were amplified by ExTaq DNA polymerase (Takara)
using the primers EGFPf plus EGFPr and DsRedf plus DsRedr
(Supplementary Table S9), respectively. The relative band in-
tensities were analyzed by computerized densitometry using
Lane and Spot Analyzer (ATTO). These experiments were re-
peated five times. Relative EGFP values derived from EGFP-
retrotransposon mRNA injections were compared with those
derived from cognate control injections by Dunnet’s multiple
Preparation of low-molecular-weight RNA and Northern blot
To obtain the low-molecular-weight RNA fraction, total RNA
solution was added with an equal amount of PEG solution (1.6
M NaCl, 13% PEG 6000), and then the supernatant was precipi-
tated using isopropanol. For Northern blot analysis of miRNAs
and gsRNAs, 20 µg of low-molecular-weight RNA was loaded
on a 15% polyacrylamide gel and electroblotted onto a Hybond
XL membrane (Amersham). Oligo DNA probes, which were
complementary to miRNAs or gsRNAs, were labeled with T4
polynucleotide kinase in the presence of [?-32P] ATP. The mem-
brane was prehybridized using PerfectHyb Plus (Sigma) for 1 h at
40°C. Hybridization was performed for 20 h at 40°C, and then
membranes were washed four times using low-stringency wash-
ing buffer (2× SSC, 0.1% SDS) at 50°C.
Enzymatic analysis of gsRNAs
Low-molecular-weight RNA (∼1 mg) from adult testes was sepa-
rated on a 15% polyacrylamide-urea gel, and then an ∼30-nt
band mainly containing gsRNAs was excised. gsRNAs were ex-
tracted from the gel slice, and eventually ∼5 ng of gsRNAs were
collected. For nuclease analyses, 1 ng of denatured gsRNAs were
incubated with 1 U/µL DNaseI (Roche) or 10 µg/mL RNaseA
(Nippon Gene) in 20 mM Tris-HCl (pH 8.4), 2 mM MgCl2, and
50 mM KCl for 1 h at 37°C. For terminal group analysis, 1 ng of
denatured gsRNAs were incubated with or without 20 U of calf
intestine alkaline phosphatase (Takara) for 1 h at 37°C and li-
gated using 10 U of T4 RNA ligase (Takara) for 1 h at 15°C.
RT–PCR of gsRNA precursors
For expression analysis of gsRNA precursors, 1 µg of total RNA
from various tissues and the testes at various ages were reverse
AC125187, AC130548, and AC153373), protein-coding genes
(?-actin and Histone H2 afx), and retrotransposons (IAP1 and
LINE1) were amplified by KOD-plus-DNA polymerase (Toyobo)
using the appropriate primers (Supplementary Table S9).
We thank Yukio Kurihara and Yuichiro Watanabe for com-
ments on the manuscript; Yuko Hoki, Jisu Park, and Toru Su-
zuki for assistance with injection experiments; Hiroyasu Fu-
ruumi, Koji Tajino, and Yasumitu Nagao for supplying mice;
and Akihiro Mori and Yoshiyuki Suzuki for discussion on sta-
tistical analysis. We also thank the members of the Sasaki labo-
Ambros, V. 2004. The function of animal microRNAs. Nature
Ambros, V., Lee, R.C., Lavanway, A., Williams, P.T., and Jewell,
D. 2003. MicroRNAs and other tiny endogenous RNAs in C.
elegans. Curr. Biol. 13: 807–818.
Aravin, A. and Tuschl, T. 2005. Identification and characteriza-
tion of small RNAs involved in RNA silencing. FEBS Lett.
Aravin, A.A., Lagos-Quintana, M., Yalcin, A., Zavolan, M.,
Marks, D., Snyder, B., Gaasterland, T., Meyer, J., and Tuschl,
T. 2003. The small RNA profile during Drosophila melano-
gaster development. Dev. Cell 5: 337–350.
Bailey, T.L. and Elkan, C. 1994. Fitting a mixture model by
expectation maximization to discover motifs in biopoly-
mers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2: 28–36.
Bartel, D.P. 2004. MicroRNAs: Genomics, biogenesis, mecha-
nism, and function. Cell 116: 281–297.
Bellve, A.R., Cavicchia, J.C., Millette, C.F., O’Brien, D.A., Bhat-
nagar, Y.M., and Dym, M. 1977. Spermatogenic cells of the
prepuberal mouse. J. Cell Biol. 74: 68–85.
Borsani, O., Zhu, J., Verslues, P.E., Sunkar, R., and Zhu, J.K.
2005. Endogenous siRNAs derived from a pair of natural
cis-antisense transcripts regulate salt tolerance in Arabidop-
sis. Cell 123: 1279–1291.
Carmell, M.A., Xuan, Z., Zhang, M.Q., and Hannon, G.J. 2002.
The Argonaute family: Tentacles that reach into RNAi, de-
velopmental control, stem cell maintenance, and tumori-
genesis. Genes & Dev. 16: 2733–2742.
Chen, P.Y., Manninga, H., Slanchev, K., Chien, M., Russo, J.J.,
Ju, J., Sheridan, R., John, B., Marks, D.S., Gaidatzis, D., et al.
2005. The developmental miRNA profiles of zebrafish as
determined by small RNA cloning. Genes & Dev. 19: 1288–
Deng, W. and Lin, H. 2002. miwi, a murine homolog of piwi,
encodes a cytoplasmic protein essential for spermatogenesis.
Dev. Cell 2: 819–830.
Du, T. and Zamore, P.D. 2005. microPrimer: The biogenesis and
function of microRNA. Development 132: 4645-4652.
Duchaine, F.T., Wohlschlegel, A.J., Kennedy, S., Bei, Y., Conte
Jr., D., Kaming, P., Brownell, D.R., Harding, S., Mitani, S.,
Ruvkun, G., et al. 2006. Functional proteomics reveals the
biochemical niche of C. elegans DCR-1 in multiple small-
RNA-mediated pathways. Cell 124: 343–354.
Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber,
K., and Tuschl, T. 2001a. Duplexes of 21-nucleotide RNAs
mediate RNA interference in cultured mammalian cells. Na-
ture 411: 494–498.
Elbashir, S.M., Lendeckel, W., and Tuschl, T. 2001b. RNA in-
terference is mediated by 21- and 22-nucleotide RNAs.
Genes & Dev. 15: 188–200.
Grishok, A., Sinskey, J.L., and Sharp, P.A. 2005. Transcriptional
silencing of a transgene by RNAi in the soma of C. elegans.
Genes & Dev. 19: 683–696.
Handel, M.A. and Eppig, J.J. 1979. Sertoli cell differentiation in
the testes of mice genetically deficient in germ cells. Biol.
Reprod. 20: 1031–1038.
He, L. and Hannon, G.J. 2004. MicroRNAs: Small RNAs with a
big role in gene regulation. Nat. Rev. Genet. 5: 522–531.
Herrera, L., Ottolenghi, C., Garcia-Oritz, J.E., Pellegrini, M.,
Manini, F., Ko, M.S., Nagaraja, R., Forabosco, A., and
Novel small RNAs from mouse germline
GENES & DEVELOPMENT1741
Schlessinger, D. 2005. Mouse ovary developmental RNA and
protein markers from gene expression profiling. Dev. Biol.
Houbaviy, H.B., Murray, M.F., and Sharp, P.A. 2003. Embryonic
stem cell-specific microRNAs. Dev. Cell 5: 351–358.
Hutvagner, G., McLachlan, J., Pasquinelli, A.E., Balint, E., Tus-
chl, T., and Zamore, P.D. 2001. A cellular function for the
RNA-interference enzyme Dicer in the maturation of the
let-7 small temporal RNA. Science 293: 834–838.
Kanellopoulou, C., Muljo, S.A., Kung, A.L., Ganesan, S., Drap-
kin, R., Jenuwein, T., Livingston, D.M., and Rajewsky, K.
2005. Dicer-deficient mouse embryonic stem cells are defec-
tive in differentiation and centromeric silencing. Genes &
Dev. 19: 489–501.
Khvorova, A., Reynolds, A., and Jayasena, S.D. 2003. Functional
siRNAs and miRNAs exhibit strand bias. Cell 115: 209–216.
Kuramochi-Miyagawa, S., Kimura, T., Yomogida, K., Kuroiwa,
A., Tadokoro, Y., Fujita, Y., Sato, M., Matsuda, Y., and Na-
kano, T. 2001. Two mouse piwi-related genes: miwi and
mili. Mech. Dev. 108: 121–133.
Kuramochi-Miyagawa, S., Kimura, T., Ijiri, T.W., Isobe, T.,
Asada, N., Fujita, Y., Ikawa, M., Iwai, N., Okabe, M., Deng,
W., et al. 2004. Mili, a mammalian member of piwi family
gene, is essential for spermatogenesis. Development 131:
Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T.
2001. Identification of novel genes coding for small ex-
pressed RNAs. Science 294: 853–858.
Lau, N.C., Lim, L.P., Weinstein, E.G., and Bartel, D.P. 2001. An
abundant class of tiny RNAs with probable regulatory roles
in Caenorhabditis elegans. Science 294: 858–862.
Lee, S.R. and Collins, K. 2006. Two classes of endogenous small
RNAs in Tetrahymena thermophila. Genes & Dev. 20: 28–
Lim, L.P., Glasner, M.E., Yekta, S., Burge, C.B., and Bartel, D.P.
2003. Vertebrate microRNA genes. Science 299: 1540.
Lippman, Z. and Martienssen, R. 2004. The role of RNA inter-
ference in heterochromatic silencing. Nature 431: 364–370.
Liu, J., Carmell, M.A., Rivas, F.V., Marsden, C.G., Thomson,
J.M., Song, J.J., Hammond, S.M., Joshua-Tor, L., and Hannon,
G.J. 2004. Argonaute2 is the catalytic engine of mammalian
RNAi. Science 305: 1437–1441.
Lu, J., Qian, J., Chen, F., Tang, X., Li, C., and Cardoso, W.V.
2005. Differential expression of components of the mi-
croRNA machinery during mouse organogenesis. Biochem.
Biophys. Res. Commun. 334: 319–323.
Lyle, R., Watanabe, D., te Vruchte, D.T., Lerchner, W., Smrzka,
O.W., Wutz, A., Schageman, J., Hahner, L., Davies, C., and
Barlow, D.P. 2000. The imprinted antisense RNA at the
Igf2R locus overlaps but does not imprint Mas1. Nat. Genet.
Ma, J.B., Yuan, Y.R., Meister, G., Peli, Y., Tuschl, T., and Patel,
D.J. 2005. Structural basis for 5?-end-specific recognition of
guide RNA by the A. fulgidus Piwi protein. Nature 434:
Maestre, J., Tchenio, T., Dhellin, O., and Heidmann, T. 1995.
mRNA retroposition in human cells: Processed pseudogene
formation. EMBO J. 14: 6333–6338.
Matranga, C., Tomari, Y., Shin, C., Bartel, D.P., and Zamore,
P.D. 2005. Passenger-strand cleavage facilitates assembly of
siRNA into Ago2-containing RNAi enzyme complexes. Cell
Mochizuki, K., Fine, N.A., Fujisawa, T., and Gorovsky, M.A.
2002. Analysis of a piwi-related gene implicates small RNAs
in genome rearrangement in Tetrahymena. Cell 110: 689–
Morris, K.V., Chan, S.W., Jacobsen, S.E., and Looney, D.J. 2004.
Small interfering RNA-induced transcriptional gene silenc-
ing in human cells. Science 305: 1289–1292.
Murchison, E.P., Partridge, J.F., Tam, O.H., Cheloufi, S., and
Hannon, G.J. 2005. Characterization of Dicer-deficient mu-
rine embryonic stem cells. Proc. Natl. Acad. Sci. 102: 12135–
Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R.
2003. Manipulating the mouse embryo. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
Paillard, L., Omilli, F., Legagneux, V., Bassez, T., Maniey, D.,
and Osborne, H.B. 1998. EDEN and EDEN-BP, a cis element
and an associated factor that mediate sequence-specific
mRNA deadenylation in Xenopus embryos. EMBO J. 17:
Park, C.W., Chen, Z., Kren, B.T., and Steer, C.J. 2004. Double-
stranded siRNA targeted to the huntingtin gene does not
induce DNA methylation. Biochem. Biophys. Res. Com-
mun. 323: 275–280.
Peaston, A.E., Evsikov, A.V., Graber, J.H., de Vries, W.N., Hol-
brook, A.E., Solter, D., and Knowles, B.B. 2004. Retrotrans-
posons regulate host genes in mouse oocytes and preimplan-
tation embryos. Dev. Cell 7: 597–606.
Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H.L., and Poet-
hig, R.S. 2004. SGS3 and SGS2/SDE1/RDR6 are required for
juvenile development and the production of trans-acting
siRNAs in Arabidopsis. Genes & Dev. 18: 2368–2379.
Pfeffer, S., Lagos-Quintana, M., and Tuschl, T. 2005. Cloning of
small RNA molecules. In Current protocols in molecular
biology (eds. R.B.F.M. Ausubel et al.), pp. 26.4.1–26.4.18.
Wiley Interscience, New York.
Schwarz, D.S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and
Zamore, P.D. 2003. Asymmetry in the assembly of the RNAi
enzyme complex. Cell 115: 199–208.
Shi, H., Djikeng, A., Tschudi, C., and Ullu, E. 2004. Argonaute
protein in the early divergent eukaryote Trypanosoma bru-
cei: Control of small interfering RNA accumulation and ret-
roposon transcript abundance. Mol. Cell. Biol. 24: 420–427.
Sijen, T. and Plasterk, R.H. 2003. Transposon silencing in the
Caenorhabditis elegans germline by natural RNAi. Nature
Sleutels, F., Zwart, R., and Barlow, D.P. 2002. The non-coding
Air RNA is required for silencing autosomal imprinted
genes. Nature 415: 810–813.
Surdej, P. and Jacobs-Lorena, M. 1998. Developmental regula-
tion of bicoid mRNA stability is mediated by the first 43
nucleotides of the 3? untranslated region. Mol. Cell. Biol. 18:
Svoboda, P., Stein, P., Hayashi, H., and Schultz, R.M. 2000.
Selective reduction of dormant maternal mRNAs in mouse
oocytes by RNA interference. Development 127: 4147–4156.
Svoboda, P., Stein, P., Anger, M., Bernstein, E., Hannon, G.J.,
and Schultz, R.M. 2004a. RNAi and expression of retrotrans-
posons MuERV-L and IAP in preimplantation mouse em-
bryos. Dev. Biol. 269: 276–285.
Svoboda, P., Stein, P., Flipowicz, W., and Schultz, R.M. 2004b.
Lack of homologous sequence-specific DNA methylation in
response to stable dsRNA expression in mouse oocytes.
Nucleic Acids Res. 32: 3601–3606.
Tomari, Y. and Zamore, P.D. 2005. Perspective: Machines for
RNAi. Genes & Dev. 19: 517–529.
van de Lagemaat, L.N., Landry, J.R., Mager, D.L., and Med-
strand, P. 2003. Transposable elements in mammals pro-
mote regulatory variation and diversification of genes with
specialized functions. Trends Genet. 19: 530–536.
Viveiros, M.M., O’Brien, M., and Eppig, J.J. 2004. Protein kinase
Watanabe et al.
1742GENES & DEVELOPMENT
C activity regulates the onset of anaphase I in mouse oo- Download full-text
cytes. Biol. Reprod. 71: 1525–1532.
Volpe, T.A., Kidner, C., Hall, I.M., Teng, G., Grewal, S.I., and
Martienssen, R.A. 2002. Regulation of heterochromatic si-
lencing and histone H3 lysine-9 methylation by RNAi. Sci-
ence 297: 1833–1837.
Yan, W., Ma, L., Stein, P., Pangas, S.A., Burns, K.H., Bai, Y.,
Schultz, R.M., and Matzuk, M.M. 2005. Mice deficient in
OAS1D display reduced fertility. Mol. Cell. Biol. 25: 4615–
Yu, Z., Raabe, T., and Hecht, N.B. 2005. MicroRNA mirn122a
reduces expression of the posttranscriptionally regulated
germ cell transition protein2 (Tnp2) messenger RNA
(mRNA) by mRNA cleavage. Biol. Reprod. 73: 427–433.
Yuan, Y.R., Pei, Y., Ma, J.B., Kuryavyi, V., Zhadina, M., Meister,
G., Chen, H.Y., Dauter, Z., Tuschl, T., and Patel, D.J. 2005.
Crystal structure of A. aeolicus Argonaute, a site-specific
DNA-guided endoribonuclease, provides insights into RISC-
mediated mRNA cleavage. Mol. Cell 19: 405–419.
Zilberman, D., Cao, X., and Jacobsen, S.E. 2003. ARGONAUTE4
control of locus-specific siRNA accumulation and DNA and
histone methylation. Science 299: 716–719.
Zuker, M. 2003. Mfold Web server for nucleic acid folding and
hybridization prediction. Nucleic Acids Res. 31: 3406–3415.
Novel small RNAs from mouse germline
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