Small non-coding RNAs, including microRNAs (miRNAs),
endogenous small interfering RNAs (endo-siRNAs) and Piwi-
interacting RNAs (piRNAs), play essential roles in mammalian
development. The function and timing of expression of these
three classes of small RNAs differ greatly. piRNAs are expressed
and play a crucial role during male gametogenesis, whereas
endo-siRNAs are essential for oocyte meiosis. By contrast,
miRNAs are ubiquitously expressed in somatic tissues and
function throughout post-implantation development.
Surprisingly, however, miRNAs are non-essential during pre-
implantation embryonic development and their function is
suppressed during oocyte meiosis. Here, we review the roles of
small non-coding RNAs during the early stages of mammalian
development, from gamete maturation through to
Key words: siRNA, MicroRNA, Stem cells, Oocyte, Embryonic
The processes of growth and differentiation are kept in balance
during the development of multicellular organisms. Post-
transcriptional control of gene expression plays a key role in this
balance by coordinating the expression of selected genes at specific
times and places. The role of post-transcriptional regulation is
particularly apparent in early mammalian development, from
maturation of the germ line to initiation of gastrulation, when
controls on mRNA localization, stability and translation are the
fundamental means of gene regulation. Indeed, from the fully
grown oocyte stage until zygotic genome activation (ZGA), the
genome is transcriptionally silent (Abe et al., 2010). Therefore, all
mRNA regulation must occur post-transcriptionally. Following
ZGA, the embryo establishes populations of early stem cells (SCs)
within the inner cell mass (ICM, the collection of cells that
eventually will form the fetus) that begin to proliferate rapidly,
ensuring that stocks of unspecialized cells are established for future
differentiation into the three germ layers. This rapid growth and the
subsequent switch from unspecialized cells into specific cell types
is a highly regulated process involving much post-transcriptional
regulation (Lu et al., 2009). Similar regulation also occurs in the
extra-embryonic tissues. This review will focus on one group of
post-transcriptional regulators, the small non-coding RNAs. These
RNAs range in size from 18 to 32 nucleotides (nt) in length and
have emerged in the past decades as major players in post-
transcriptional regulation across many, if not most, multicellular
Classes and biogenesis of mammalian small RNAs
Three major classes of functional small non-coding RNAs have
been found in mammals: microRNAs (miRNAs), endogenous
small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs
(piRNAs) (Babiarz and Blelloch, 2009; Kim et al., 2009; Thomson
and Lin, 2009). These classes differ in their biogenesis, i.e. their
maturation from transcribed forms to the active form of the RNA
miRNAs can be divided into two subclasses: canonical and non-
canonical miRNAs. Canonical miRNAs are initially transcribed as
long RNAs that contain hairpins (Fig. 1A). The 60-75 nt hairpins
are recognized by the RNA-binding protein Dgcr8 (DiGeorge
syndrome critical region 8), which directs the RNase III enzyme
Drosha to cleave the base of the hairpin (Denli et al., 2004;
Gregory et al., 2004; Han et al., 2004; Han et al., 2006; Landthaler
et al., 2004; Lee et al., 2003). Following cleavage by the Drosha-
Dgcr8 complex, also called the microprocessor, the released
hairpin is transported to the cytoplasm, where Dicer, another
RNase III enzyme, then cleaves it into a single short 18-25 nt
dsRNA (Bernstein et al., 2001; Hutvagner et al., 2001; Ketting et
al., 2001; Knight and Bass, 2001). Non-canonical miRNAs bypass
processing by the microprocessor by using other endonucleases or
by direct transcription of a short hairpin. The resulting pre-
miRNAs are then exported from the nucleus and cleaved once by
Dicer (Babiarz et al., 2008; Okamura et al., 2007; Ruby et al.,
By contrast, siRNAs are derived from long dsRNAs (Fig. 1B) in
the form of either sense or antisense RNA pairs or as long hairpins,
which are then directly processed by Dicer consecutively along the
dsRNA to produce multiple siRNAs (Chung et al., 2008; Czech et
al., 2008; Ghildiyal et al., 2008; Kawamura et al., 2008; Okamura
et al., 2008a; Okamura et al., 2008b). Therefore, canonical
miRNAs, non-canonical miRNAs and endo-siRNAs all involve
Dicer processing and are ~21 nt in length. Furthermore, in all three
cases, one strand of the Dicer product associates with an Argonaute
protein (Ago 1-4 in mammals; also known as Eif2c1-4) to form the
active RISC (RNA-induced silencing complex, Fig. 1D)
(Filipowicz, 2005). These ribonucleoprotein complexes are able to
bind to and control the levels and translation of their target mRNAs:
if the match between the small RNA and its target is perfect, the
target is cleaved; if not, the mRNA is destabilized through as yet
unresolved mechanisms (Doench et al., 2003; Fabian et al., 2010;
Zeng et al., 2003).
The piRNAs differ from the miRNAs and endo-siRNAs in that
they do not require Dicer for their processing (Houwing et al.,
2007; Vagin et al., 2006). Indeed, how piRNAs are produced and
their mechanism of action remains poorly characterized (for a
review, see Klattenhoff and Theurkauf, 2008). piRNAs are 25-32
nt in length, and are expressed predominantly in the germline in
mammals (Aravin et al., 2006; Grivna et al., 2006; Watanabe et al.,
2006). They are defined by their interaction with the Piwi proteins,
a distinct family of Argonaute proteins (including Miwi, Miwi2
Development 138, 1653-1661 (2011) doi:10.1242/dev.056234
© 2011. Published by The Company of Biologists Ltd
Small RNAs in early mammalian development: from gametes
Nayoung Suh and Robert Blelloch*
The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research,
Center for Reproductive Sciences, and Department of Urology, University of
California San Francisco, San Francisco, CA 94143, USA.
*Author for correspondence (Blellochr@stemcell.ucsf.edu)
and Mili in mouse; also known as Piwil1, Piwil4 and Piwil2,
respectively). piRNAs are generated from long single-stranded
RNA precursors that are often encoded by complex and repetitive
intergenic sequences. One proposed model for their biogenesis is
the ‘ping-pong mechanism’ (Fig. 1C) (Aravin et al., 2007;
Brennecke et al., 2007; Gunawardane et al., 2007). In this model,
the Argonaute protein Mili cleaves the primary piRNA to define
the 5? end of piRNAs, which is subsequently recognized by Miwi2.
Miwi2 then cleaves the other strand of the precursor, thereby
generating a 5? end of the piRNA that can then bind to Mili, thus
forming a positive amplification loop. Many of the details of this
model remain to be uncovered. Furthermore, the ping-pong model
is likely to explain the biogenesis of only a subset of mammalian
piRNAs, those that are derived from repetitive sequences, such as
transposons, and that are associated with Miwi2 and Mili in the
early stages of spermatogenesis. The mechanism of biogenesis of
piRNAs derived from complex intergenic sequences, associated
with Miwi and Mili in the later stages of spermatogenesis, is
Small RNAs in gametes
Most animals, including vertebrates, reproduce sexually and have
the ability to form gametes. The two types of gametes, the egg and
the sperm, arise from immature germ cells, undergo extensive
differentiation and ultimately fuse to create their progeny. One
fascinating aspect of this process is that, upon fertilization, the
highly specialized sperm and egg unite to produce the zygote,
which is totipotent, having the potential to produce all the cells of
the body. This switch from a singular function to a totipotent cell
involves massive molecular rewiring (Hemberger et al., 2009).
Based on recent findings, small RNAs are likely to play a very
important role during this transition.
Small RNAs in spermatogenesis
Spermatogenesis is a highly regulated process, during which
diploid spermatogonia differentiate into haploid spermatozoa
within the seminiferous epithelium of the testis. During the course
of differentiation into sperm, numerous mRNAs are regulated
post-transcriptionally (Lee et al., 2009). Recent studies using both
Development 138 (9)
A miRNA pathway
D Effector pathway
B endo-siRNA pathway
C piRNA pathway
Translational repression or
Fig. 1. Biogenesis of microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs).
(A)Canonical miRNAs are processed from long primary miRNAs (pri-miRNAs) into short hairpin precursor miRNAs (pre-miRNAs) by the
microprocessor, a complex consisting of the RNA binding protein Dgcr8 and the RNase III enzyme Drosha. By contrast, non-canonical miRNAs
are transcribed directly as short hairpins (shRNAs) or derive from introns that can refold into shRNAs (mirtrons). (B)Precursors of endo-siRNAs
are derived from long stem-loop structures (inverted repeat), opposing strand transcription (cis endo-siRNAs), or gene-pseudogene pairs (trans
endo-siRNAs). Both miRNAs and endo-siRNAs are then processed by the RNase III enzyme Dicer to produce double-stranded RNAs of ~21
nucleotides. (C)piRNAs are processed from single-stranded RNA precursors that are often encoded by intergenic repetitive elements or
transposons. The mechanisms that drive piRNA biogenesis are not well understood, although a ‘ping-pong mechanism’ has been described
for a subset of piRNAs. In this model, Mili cleaves the primary piRNA, which is subsequently recognized by Miwi2. Miwi2 cleaves the other
strand of the precursor that can then bind to Mili, thus forming a positive amplification loop. (D)Following their processing, miRNAs and
endo-siRNAs are assembled into ribonucleoprotein (RNP) complexes called RNA-induced silencing complexes (RISCs). The key components of
RISCs are proteins of the Argonaute (Ago) family. In mammals, four Ago proteins (Ago 1-4) function in miRNA repression but only Ago2
functions in siRNA repression. The fate of piRNAs is unknown. On the DNA, blue represents the positive strand and red represents the
Development 138 (9)
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