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P o s t- t R A N s c R i P t i o N A l c o N t R o l

Biogenesis of small RNAs in animals
V. Narry Kim*, Jinju Han* and Mikiko C. Siomi‡

Abstract | Small RNAs of 20–30 nucleotides can target both chromatin and transcripts, andthereby keep both the genome and the transcriptome under extensive surveillance. Recent
progress in high-throughput sequencing has uncovered an astounding landscape of small
RNAs in eukaryotic cells. Various small RNAs of distinctive characteristics have been foundand can be classified into three classes based on their biogenesis mechanism and the typeof Argonaute protein that they are associated with: microRNAs (miRNAs), endogenous smallinterfering RNAs (endo-siRNAs or esiRNAs) and Piwi-interacting RNAs (piRNAs). This Review
summarizes our current knowledge of how these intriguing molecules are generated inanimal cells.
Heterochromatin
Highly condensed regions of
the genome in whichtranscription is generally
limited.

RNase III-type protein
An endonuclease that cleaves
double-stranded RNAs andcreates 5′-phosphate and3′-hydroxyl termini, leaving
2-nucleotide 3′ overhangs.

*School of Biological
Sciences and Center for
National Creative Research,
Seoul National University,
Seoul, 151‑742, Korea.
‡
Keio University School of
Medicine, 35 Shinanomachi,
Shinjuku‑ku, Tokyo
160‑8582, and the Japan
Science and Technology
Agency (JST), Core Research
for Evolutionary Scienceand Technology (CREST),
Saitama 332‑0012, Japan.
Correspondence to V.N.K.
and M.C.S.
e‑mails: narrykim@snu.ac.kr;
siomim@sc.itc.keio.ac.jp
doi:10.1038/nrm2632

The first small RNA, lin‑4, was discovered in 1993 bygenetic screens in nematode worms1,2. The number ofknown small RNAs has since expanded substantially,
mainly as a result of the cloning and sequencing of size‑
fractionated RNAs3–5. The recent development of deep‑
sequencing technologies6,7 and computational prediction
methods8–11 has accelerated the discovery of less abun‑dant small RNAs. The functions of small RNAs range
from heterochromatin formation to mRNA destabilizationand translational control12,13. Through such extensive
patrolling across the genome and transcriptome, small
RNAs are involved in almost every biological process,including developmental timing, cell differentiation, cellproliferation, cell death, metabolic control, transposon
silencing and antiviral defence.
‘Small RNA’ is a rather arbitrary term, because it waspreviously used for other non‑coding RNAs, such as small
nuclear RNAs (snRNAs) and transfer RNAs (tRNAs).
Bacterial short regulatory RNAs have also been referred
to as small RNAs, but they are not related to eukaryotic
small RNAs. What distinguishes and defines eukaryo‑
tic small RNAs in the RNA silencing pathway is theirlimited size (~20–30 nucleotides (nt)) and their associa‑
tion with Argonaute (Ago)‑family proteins (BOX 1; TABLE 1).
The Ago family can be grouped further into two clades:
the Ago subfamily and the Piwi subfamily. At least threeclasses of small RNAs are encoded in our genome, basedon their biogenesis mechanism and the type of Ago pro‑tein that they are associated with: microRNAs (miRNAs),
endogenous small interfering RNAs (endo‑siRNAs or
esiRNAs) and Piwi‑interacting RNAs (piRNAs). It should
be noted, however, that the recent discoveries of numerous
non‑canonical small RNAs have blurred the boundaries
between the classes.

The best understood among the three classes,
miRNAs are generated from local hairpin structures by
the action of two RNase III-type proteins, Drosha and Dicer(BOX 2). Mature miRNAs of ~22 nt are then bound by
Ago‑subfamily proteins. miRNAs target mRNAs andthereby function as post‑transcriptional regulators.
The longest of the three classes, piRNAs (24–31 nt inlength) are associated with Piwi‑subfamily proteins.
Intriguingly, the biogenesis of piRNAs does not dependon Dicer 14. piRNAs are highly abundant in germ cells
and at least some of them are involved in transposon
silencing through heterochromatin formation or RNA
destabilization. endo‑siRNAs have been studied mostly
in Drosophila melanogaster, although they have also
been found in mouse oocytes and embryonic stem (ES)cells15–17. Despite their similarity with miRNAs in termsof their association with Ago‑subfamily proteins, endo‑
siRNAs differ from miRNAs in that they are derived
from long double‑stranded RNAs (dsRNAs) and are
dependent only on Dicer and not on Drosha18–20. They
are also slightly shorter (~21 nt) than miRNAs. At least
some of the endo‑siRNAs have been shown to function
as post‑transcriptional regulators that target RNAs.
This Review summarizes our current knowledge ofthe biogenesis pathways of small RNAs. The focus of the
Review will remain on animal small RNAs, mainly inmammals and flies. Several excellent reviews on small
RNAs in plants and yeast are available elsewhere21–24.

microRNA biogenesis
miRNAs are single‑stranded RNAs (ssRNAs) of ~22 nt
in length that are generated from endogenous hair‑pin‑shaped transcripts25. miRNAs function as guidemolecules in post‑transcriptional gene regulation by

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REVIEWS
Box 1 | Argonaute proteins and their associated small RNAs
The Argonaute (Ago)
a
PAZ
MID
PIWI
family can be classified
NC
into two subclades: theAgo subfamily and thebCatalytic site
PIWI
Piwi subfamily (TABLE 1).
The Ago proteins are
expressed ubiquitously,interact with microRNAs(miRNAs) or small
MID
PAZ
interfering RNAs(siRNAs), and function as
3′ end ofpost-transcriptionalguide strand
5′ end ofregulators. The Piwi
guide strand
proteins are abundantly
expressed in germ cells and function in transposon silencing, together with
Nature Reviews | Molecular Cell Biology
Piwi-interacting RNAs (piRNAs).
Ago-family proteins are composed of three characteristic domains: the PAZ, MID
and PIWI domains (see the figure, part a). The PAZ domain serves as a docking site forthe 3′ end of small RNA181–184, whereas the MID domain anchors the 5′ terminal
nucleotide183–187 (see the figure, part b). Recent studies have determined the structureof Thermus thermophilus Ago with a guide strand and target strand duplex183,184. The
PIWI domain has a structure that is similar to RNase H, which cuts the RNA strand of
an RNA–DNA hybrid. Indeed, the PIWI domain of some Ago proteins can cleave thetarget RNA bound to small RNA: this is called slicer activity. Of the four human Agoproteins (AGO1–4; also known as EIF2C1–4), only AGO2 has slicer activity, whereas in
Drosophila melanogaster all Ago and Piwi proteins possess slicer activity. Apart fromthe endonucleolytic cleavage that is mediated by the PIWI domain, the Ago proteins
can induce translational repression and exonucleolytic mRNA decay throughinteraction with other protein factors13.
The fly Piwi proteins Aubergine (AUB) and AGO3 can cleave target mRNAs,
resulting in the silencing of retrotransposons, other intergenic repetitive elements
and protein-coding genes, such as Stellate (also known as SteXh)145,148. PIWI might
function differently from AUB and AGO3, as it associates with chromatin andinteracts with HP1a (heterochromatin protein 1a), which is involved inheterochromatin formation. Thus, PIWI might contribute to the epigenetic control of
the fly genome188. Consistently, AUB and AGO3 are localized in the cytoplasm,
whereas PIWI is found in the nucleus145–148. The expression patterns are also different;
PIWI, but not AUB and AGO3, was detected in the soma of ovaries, indicatingthat PIWI has a function outside of germ cells. Epigenetic changes were observedin Mili (also known as Piwil2) and Miwi2 (also known as Piwil4) knockout mice,indicating that mammalian Piwi homologues function in a similar way to fly PIWI inheterochromatin control156.
Derepression of transposons was shown in flies that were deficient in Dicer 2 orAgo2 and in S2 cells that lack Dicer 2 (REFs 19,20,170,171), which suggests that
endogenous siRNAs (endo-siRNAs) as well as piRNAs can defend against nucleic
acid-based parasites. Some piRNAs and endo-siRNAs are complementary toprotein-coding genes and they might function in trans to suppress protein-coding gene
expression, probably through mRNA cleavage18,20. Figure part b is reproduced, withpermission, from Nature REF. 183  (2008) Macmillan Publishers Ltd. All rights reserved.

Bilaterian
An animal that has a front,a back, an upside and adownside (bilateral symmetry).

Paralogue
A gene or protein with a highly
similar sequence to anotherthat is encoded in the same
genome.

base‑pairing with the target mRNAs, usually in the
3′ untranslated region (uTR). Binding of a miRNAto the target mRNA typically leads to translational
repression and exonucleolytic mRNA decay, althoughhighly complementary targets can be cleaved endo‑
nucleolytically. other types of regulation, such astranslational activation13 and heterochromatin for‑mation26, have also been described. over one‑thirdof human genes are predicted to be directly targetedby miRNAs. Consequently, the unique combinationof miRNAs in each cell type determines the use of
thousands of mRNAs.

miRNA genes and their transcription. At present, themiRNA database contains 154 Caenorhabditis elegans,
152 D. melanogaster, 337 Danio rerio (zebrafish), 475 Gallus
gallus (chicken), 695 human and 187 Arabidopsis thal‑
iana miRNAs. miRNAs are even present in simple multi‑
cellular organisms, such as poriferans (sponges) andcnidarians (starlet sea anemone)27. Many of the bilateriananimal miRNAs are phylogenetically conserved; ~55%
of C. elegans miRNAs have homologues in humans,
which indicates that miRNAs have had important roles
throughout animal evolution28. Animal miRNAs seemto have evolved separately from those in plants because
their sequences, precursor structure and biogenesis
mechanisms are distinct from those in plants29,30.
Most mammalian miRNA genes have multiple iso‑forms (paralogues) that are probably the result of gene
duplications. for instance, the human genome has
12 loci for let‑7‑family miRNAs. Paralogues often haveidentical sequences at nucleotide positions 2–7 relative
to the 5′ end of the miRNA. Because these six nucleo‑
tides (called seed) are crucial in base pairing with thetarget mRNA, the paralogues are thought to act redun‑
dantly. However, because the 3′ sequences of miRNAs
also contribute to target binding and because the expres‑sion patterns of these sister miRNAs are often different
from each other, members of the same seed family might
have distinct roles in vivo31.
Approximately 50% of mammalian miRNA lociare found in close proximity to other miRNAs. Theseclustered miRNAs are transcribed from a single polycistronic transcription unit (Tu)32, although there may
be exceptional cases in which individual miRNAs arederived from separate gene promoters. Some miRNAs
are generated from non‑coding Tus, whereas others are
encoded in protein‑coding Tus (FIG. 1). Approximately
40% of miRNA loci are located in the intronic region ofnon‑coding transcripts, whereas ~10% are placed in theexonic region of non‑coding Tus. miRNAs in protein‑
coding Tus are usually found in intronic regions, which
account for ~40% of all miRNA loci. Some ‘mixed’
miRNA genes can be assigned to either intronic or
exonic miRNA groups depending on the alternative
splicing patterns.
The transcription of most miRNA genes is medi‑
ated by RNA polymerase II (Pol II)33,34, although aminor group of miRNAs that are associated with Alu
repeats can be transcribed by Pol III35. A range of
Pol II‑associated transcription factors control miRNA
gene transcription36. Thus, Pol II‑dependent transcrip‑tion allows miRNA genes to be elaborately regulated in
specific conditions and cell types.
Nuclear processing by Drosha. The primary transcripts(pri‑miRNAs) that are generated by Pol II are usu‑
ally several kilobases long and contain local stem-loop
structures (FIG. 2a). The first step of miRNA maturation
is cleavage at the stem of the hairpin structure, which
releases a small hairpin that is termed a pre‑miRNA32.
This reaction takes place in the nucleus by the nuclear
RNase III‑type protein Drosha 37. Drosha requires
a cofactor, the DiGeorge syndrome critical region

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Table 1 | types of small RNA-associated Argonaute proteins, and the origin and mechanism of action of small RNAs
Subfamily

ago-family proteinclass of small rNa*

length of small rNa

origin of small rNa‡

Mechanism of action

AGO1–4miRNA

21–23 nt

miRNA genes

Translational repression,mRNA degradation,
mRNA cleavage andheterochromatin
formation?

endo-siRNA§

21–22 nt

Intergenic repetitiveelements, pseudogenes
and endo-siRNA clusters

mRNA cleavage?

MILI (PIWIL2 inhumans)Pre-pachytene piRNAand pachytene piRNA

24–28 nt

Transposons and piRNA
clusters

Heterochromatin
formation (DNAmethylation)

MIWI (PIWIL1 inhumans)Pachytene piRNA

29–31 nt

piRNA clusters?

MIWI2 (PIWIL4 inhumans)Pre-pachytene piRNA

27–29 nt

Transposons and piRNA
clusters

Heterochromatin
formation (DNAmethylation)(PIWIL3 in humans)

?

?

?

?

AGO1

miRNA

21–23 nt

miRNA genes

Translational repression
and mRNA degradation

AGO2

endo-siRNA~21 ntTransposons, mRNAs andrepeats

RNA cleavage

Mammals
Ago

Piwi

Drosophila melanogaster
Ago

Piwi

exo-siRNA~21 ntViral genome

Viral RNA cleavage

AUB

piRNA

23–27 nt

Transposons, repeats,piRNA clusters and Su(Ste)locus

RNA cleavage

AGO3

piRNA

24–27 nt

Transposons and repeats(unknown in testis)

RNA cleavage

PIWI

piRNA

24–29 nt

Transposons, repeats andpiRNA clusters

Heterochromatin
formation?

endo-siRNA~21 ntOuter centromeric repeats, Heterochromatin
mating-type locus andformation
subtelomeric regions

miRNA

20–24 nt

miRNA genes

mRNA cleavage andtranslational repression

endo-siRNA (tasiRNA
including TAS3)

21 nt

TAS genes

mRNA cleavage

exo-siRNA

20–22 nt

Viral genome

Viral RNA cleavage

AGO4 and AGO6

rasiRNA

24 nt

Transposons and repetitiveelements

Heterochromatin
formation

AGO7

miR-390

21 nt

miRNA gene

Cleavage of TAS3 RNA

Schizosaccharomyces pombe
Ago

Ago1

Arabidopsis thaliana||
Ago

AGO1

*Small RNAs that are the main partners of a given Ago protein are listed. ‡miRNAs, as a class, are expressed in all cell types, whereas endo-siRNAs and piRNAs are
expressed abundantly in germ cells and contribute to germline development. §So far, only AGO2 has been shown to be required for endo-siRNAs. ||Plants have ten
Ago proteins, but only those with known small RNA partners are shown. Ago, Argonaute; AUB, Aubergine; endo-siRNA, endogenous small interfering RNA;
exo-siRNA, exogenous small interfering RNA; miRNA, microRNA; nt, nucleotide; piRNA, Piwi-interacting RNA; rasiRNA, repeat-associated siRNA; Su(Ste),Suppressor of Stellate; TAS, tasi gene; tasiRNA, trans-acting siRNA.

Polycistronic transcription
unit
An RNA transcript that
includes regions that represent
multiple gene products.

gene 8 (DGCR8) protein in humans (Pasha in D. mela‑nogaster and C. elegans)38–41. Together with DGCR8(or Pasha), Drosha forms a large complex known as
the Microprocessor complex, which is ~500 kDa in
D. melanogaster 39 and ~650 kDa in humans38,40. Mouse
ES cells that are deficient in the Dgcr8 gene fail to pro‑
duce miRNAs and manifest defects in proliferation anddifferentiation, thereby confirming the essential roleof DGCR8 in the miRNA pathway and the importance ofmiRNAs in ES‑cell function42. Drosha and DGCR8 areconserved only in animals43–45.
How does the Microprocessor complex recognize its
substrates? A typical metazoan pri‑miRNA consists of astem of ~33 base pairs (bp), a terminal loop and flanking

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Box 2 | RNase iii proteins and their mechanism of actionaHuman Drosha

P-richRS-richRIIIDa RIIIDb dsRBD(1,374 aa)

WW

Human DGCR8Human Dicer

DEAD

dsRBD
(773 aa)

Helicase DUF283

PAZ

RIIIDaRIIIDb dsRBD(1,922 aa)bc
RIIIDbRIIIDaDroshaRIIIDbRIIIDa5′~11 bp3′

65 Å
Platform

DGCR8

PAZ

Two types of RNase III are found in animals: Drosha and Dicer. Both proteins have two
NatureRNA-binding
Reviews | Molecular
Biologytandem RNase III domains (RIIIDs) and a double-stranded
domainCell(dsRBD;see the figure, part a). Two RIIIDs interact with each other to make an intramoleculardimer in which the two catalytic sites are located closely to each other. The first RIIID
cuts the 3′ strand of dsRNA, whereas the second RIIID cleaves the 5′ strand, generating
a 2-nucleotide (nt) 3′ overhang38,189,190 (see the figure, parts b,c).
Drosha is a nuclear protein of 130–160 kDa. The dsRBD is necessary for activity,
although it is not sufficient to bind a primary transcript (pri-miRNA). To provide theRNA-binding activity, Drosha interacts with a dsRNA-binding protein, DiGeorge
syndrome critical region gene 8 (DGCR8; Pasha in Drosophila melanogaster andCaenorhabditis elegans), through its middle region (this Drosha–DGCR8 complex is
known as the Microprocessor; see the figure, part b). The amino-terminal region contains
the nuclear localization signal, which is dispensable for cleavage activity in vitro38,191.
DGCR8 and Pasha are ~120 kDa and localize to the nucleoplasm and the nucleolus177,191.
They contain two dsRBDs that recognize the ssRNA–dsRNA junction of a substrate46.
DGCR8 and Pasha interact with Drosha through their C-terminal domain191. A structureof the partial human DGCR8 protein that contains the two dsRBDs and a part of the
carboxy-terminal domain showed that the two dsRBDs are arranged with pseudo
two-fold symmetry and that the C-terminal helix is closely packed against the two
dsRBDs192. The middle domain of DGCR8 is required for haem binding, oligomerizationand pri-miRNA processing191,193.
Dicer homologues are cytoplasmic RNase III proteins of ~200 kDa. The middle region of
Dicer contains a PAZ domain, which binds to the 3′ protruding end of RNAs181,182,194,195.
Structural studies on the Giardia intestinalis Dicer protein show that the processing centre
made of the two RIIIDs is connected to the PAZ domain by a long, positively charged
helix196. Based on the structure (see the figure, part c), it is predicted that the 3′ end of adsRNA is docked to the PAZ domain and the RNA stem interacts with the flat, positively
charged extension to reach the catalytic centre. The distance between the PAZ domain
and the catalytic site (65 Å) approximately matches the length of the product of
G. intestinalis Dicer (25 nt). The DEAD-box RNA helicase domain is not necessary for
Dicer activity in vitro78, and its role remains unknown. Figure part c is reproduced, withpermission, from REF. 196  (2006) American Association for the Advancement of Science.

ssRNA segments. DGCR8 interacts with pri‑miRNAs
through the ssRNA segments and the stem of ~33 bp,
and assists Drosha to cleave the substrate ~11 bp awayfrom the ssRNA–dsRNA junction46,47.

Recent studies show that pri‑miRNA processing
might be a co‑transcriptional process48–50. The initialmodel was based on the finding that Drosha process‑ing of intronic miRNA precedes the splicing of a hostintron48 (FIG. 2b). Interestingly, the cleavage of the intron
by Drosha does not impair splicing 48. This is consist‑ent with a previous ‘exon‑tethering’ model 51, whichsuggested that the exons of Pol II transcripts are co‑transcriptionally assembled into the spliceosome. Thus,
Drosha processing might take place after the transcript
is tied to the splicing commitment complex (also known
as the early spliceosome complex), but before the intron
is excised. Thus, cropping and splicing might be highly
coordinated co‑transcriptional processes. Supporting
this, pri‑miRNA and Drosha localize to the tran‑
scription sites50 and pri‑miRNAs are enriched in thechromatin‑associated nuclear fraction49,50. Chromatin
immunoprecipitation and nuclear run‑on assays haveprovided further evidence that pri‑miRNA processing
is a co‑transcriptional process49,50. The nuclear exosome(which comprises 3′→5′ exonucleases) and XRN2 (a5′→3′ exonuclease) also associate with the intronic
miRNA hairpin region and promote the degradationof the fragments49,50.
When the miRNA hairpin is located in the exonic
region, Drosha processing can destabilize the transcript
and reduces protein synthesis from it 52. It was recently
shown that Drosha can cleave not only pri‑miRNAs but
also mRNAs that contain long hairpins52. Drosha nega‑tively regulates its own cofactor, DGCR8, by cleaving
the hairpins in the second exon of the DGCR8 mRNA52.
It would be interesting to establish how widespread thistype of mRNA stability control is.
Apart from canonical intronic miRNAs, small
groups of miRNA‑like RNAs have been discovered in
introns in flies and mammals53–55. These small RNAs
are embedded in short introns, and their biogenesis
does not require Drosha processing (FIG. 2c). following
the completion of splicing, the branch point of thelariat‑shaped intron is resolved and the debranched
intron forms a hairpin structure that resembles pre‑miRNA. Some precursors (mirtrons) contain extended
tails at either the 5′ or 3′ end, which therefore require
exonucleolytic trimming in order to become a sub‑strate for nuclear export (FIG. 2c). Small RNAs that arederived from other non‑coding RNAs, such as tRNA17
or small nucleolar RNA (snoRNA)56, have also been
described. Therefore, it is becoming clear that multi‑
ple non‑canonical pathways can feed pre‑miRNAs into
the miRNA pathway through Drosha‑independent
processes.
Nuclear export by exportin 5. following nuclear process‑ing, pre‑miRNAs are exported to the cytoplasm57. Exportof pre‑miRNAs is mediated by exportin 5 (EXP5),
which is a member of the nuclear transport receptor
family 58–60. Although EXP5 was originally known as aminor export factor for tRNAs61,62, the major cargos of
EXP5 turned out to be pre‑miRNAs58–60. As with othernuclear transport receptors, EXP5 binds cooperatively
to its cargo and the GTP‑bound form of the cofactor

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REVIEWSa Non-coding TU with intronic miRNA

miR-15a~16-1

DLEU2

miR-155b Non-coding TU with exonic miRNABIC

c Coding TU with intronic miRNA

miR-25~93~106b

MCM7

d Coding TU with exonic miRNA

miR-985

CACNG8Figure 1 | genomic location and gene structure of mirNas. MicroRNAs (miRNAs) can
be categorized into four groups according to their genomic locations relative to exon
Nature Reviews | Molecular Cell Biology
and intron positions. a | Intronic miRNAs in non-coding transcripts, such as themiR-15a~16-1 cluster. The miR-15a~16-1 cluster is found in the intron of a well-defined
non-coding RNA gene, DLEU2 (REF. 197). b | Exonic miRNAs in non-coding transcripts.
miR-155 was found in a previously defined non-coding RNA gene, BIC198. c | Intronic
miRNAs in protein-coding transcripts. Shown here as an example is the miR-25~93~106bcluster, which is embedded in the intron of the DNA replication licensing factor MCM7transcript. d | Exonic miRNAs in protein-coding transcripts. The miR-985 hairpin isfound in the last exon of CACNG8 mRNA. The hairpins represent miRNA stem-loops.
Blue boxes indicate the protein-coding regions. This figure is roughly to scale.
TU, transcription unit.

Stem-loop structure
A lollipop-shaped structurethat is formed when asingle-stranded nucleic acidmolecule loops back on itself
to form a complementary
double helix (stem) topped bya loop.

MirtronA microRNA that is generated
from a short spliced intron
without Drosha-mediatedcleavage.

dsRBD(Double-stranded-RNA-binding
domain). A protein domainthat binds to the A-form
double-stranded RNA helix.
Proteins that contain a dsRBD
function in RNA localization,
editing, translational repression
and post-transcriptional genesilencing.

Ran in the nucleus, and releases the cargo following thehydrolysis of GTP in the cytoplasm. EXP5 recognizes
the >14‑bp dsRNA stem along with a short 3′ overhang(1–8 nt)58,63–65.
Cytoplasmic processing by Dicer. following export fromthe nucleus, pre‑miRNAs are cleaved near the terminal
loop by Dicer, releasing ~22‑nt miRNA duplexes66–70(FIG. 2a). Thus, Drosha predetermines mature miRNA
sequences by generating one end of the mature miRNA,
whereas the other end is created by Dicer, which meas‑ures ~22 nt from the pre‑existing terminus of the pre‑miRNA (BOX 2). Dicer is a highly conserved protein thatis found in almost all eukaryotic organisms, includ‑ing Schizosaccharomyces pombe, plants and animals.
Some organisms contain multiple Dicer homologues,
whereby different Dicer isotypes have distinct roles71,72.
for instance, D. melanogaster Dicer 1 is required formiRNA biogenesis, whereas Dicer 2 functions in siRNA
production71.
Dicer associates with dsRNA‑binding proteins.
D. melanogaster Dicer 1 requires loquacious (loQS; also
known as R3D1), which contains three dsRNA‑binding

domains ( dsRBDs ) for pre‑miRNA processing 73–75.
Human Dicer interacts with two closely related pro‑teins, TRBP (TAR RNA‑binding protein; also known
as TARBP2)76,77 and PACT (also known as PRKRA)78.
Although neither TRBP nor PACT are required forprocessing activity itself, they seem to contribute toformation of the RNA‑induced silencing complex(RISC; see below)76–78. The detailed biochemical rolesof these proteins remain to be determined. Earlier
studies reported that TRBP functions as a negative
regulator of dsRNA‑dependent protein kinase (PKR;
also known as EIf2AK2), whereas PACT stimulates
PKR79. At present, it is unclear whether there is any
functional connection between the miRNA pathway
and PKR signalling.
Argonaute loading. following Dicer cleavage, the result‑ing ~22‑nt RNA duplex is loaded onto an Ago protein
so as to generate the effector complex, RISC. one strandof the ~22‑nt RNA duplex remains in Ago as a mature
miRNA (the guide strand or miRNA), whereas the otherstrand (the passenger strand or miRNA*) is degraded.
Studies on siRNA duplexes indicate that the relative
thermodynamic stability of the two ends of the duplex
determines which strand is to be selected80–82. The
strand with relatively unstable base pairs at the 5′ end
typically survives (for example, a Gu pair comparedwith a GC pair)81,82. Thermodynamic stability profilingstudies on miRNA precursors suggested that the same
rule might apply to most, although not all, miRNAs46,82.
Because strand selection is often not a stringent process,some hairpins produce miRNAs from both strands atcomparable frequencies.
Dicer, TRBP (and/or PACT) and Ago proteins con‑tribute to RISC assembly by forming a RISC loading
complex (RlC) in humans (whereas in flies, RlC com‑prises Dicer 1, loQS and AGo1)76,83–86. Although it iscurrently unknown how RlC binds to RNA and facilitates
Ago loading, evidence suggests that the miRNA duplex
is released from Dicer after cleavage and that the stableend of the RNA duplex is bound to TRBP in the RlC,
whereas the other end interacts with the Ago protein86,87
The mechanistic details of strand selection and RISC
assembly have been best delineated with synthetic
RNA duplexes in D. melanogaster extracts86,88. R2D2,
a protein with two dsRBDs, senses the thermodynamic
inequalities. R2D2 forms a stable heterodimeric com‑plex with Dicer 2, binds to the more stable end of the
RNA duplex, and orientates AGo2, on the RNA duplex.
It was shown that the endonucleolytic enzymatic activ‑
ity (slicer activity) of Ago protein is responsible for theremoval of the passenger strand of siRNA duplexes andsome miRNA duplexes89–92. However, most miRNA
duplexes (unlike siRNA duplexes) contain mismatches
in the middle, and some Ago proteins (for example,
AGo1, AGo3 and AGo4 in humans; also known as
EIf2C1, EIf2C3 and EIf2C4, respectively) lack slicer
activity, which prevents the cleavage of the passenger
strand. An RNA helicase activity is thought to mediate
the unwinding and removal of the unselected strand of themiRNA duplex.

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REVIEWSa Biogenesis of canonical miRNA
miRNA geneb Canonical intronic miRNA
Microprocessor(Drosha–DGCR8)

Pol IIPol II

Transcription

DNA

Commitmentcomplexpri-miRNA

Cropping

DroshaPol II

pre-miRNA

AAAAA

m7G

DGCR8
(Pasha in flies)

Spliceosome

Cropping
DicingSplicing

Mature miRNA

Mature mRNA

pre-miRNA
Exportin 5–RanGTP

c Non-canonical intronic small RNA (mirtron)

ExportNucleuspre-mRNA

Spliceosome

Cytoplasm
Dicer(Dicer 1 in flies)

AGO1–4(AGO1 in flies)

Splicing
Mature mRNATRBP or PACT(LOQS in flies)

Debranching

Dicing
TRBP or PACT(LOQS in flies)

Branched pre-mirtron(excised intron)

or
Dicer
(Dicer 1 in flies)

AGO1–4(AGO1 in flies)

Trimming
pre-miRNA

Loading
Dicing
AGO1–4(AGO1 in flies)

miRNA

Mature miRNA

Figure 2 | mirNa biogenesis pathway. a | Canonical microRNA (miRNA) genes are transcribed by RNA polymerase II
Nature Reviews | Molecular Cell Biology(Pol II) to generate the primary transcripts (pri-miRNAs). The initiation step (cropping) is mediated by the
Drosha–DiGeorge syndrome critical region gene 8 (DGCR8; Pasha in Drosophila melanogaster and Caenorhabditis
elegans) complex (also known as the Microprocessor complex) that generates ~65 nucleotide (nt) pre-miRNAs.
Pre-miRNA has a short stem plus a ~2-nt 3′ overhang, which is recognized by the nuclear export factor exportin 5 (EXP5).
On export from the nucleus, the cytoplasmic RNase III Dicer catalyses the second processing (dicing) step to produce
miRNA duplexes. Dicer, TRBP (TAR RNA-binding protein; also known as TARBP2) or PACT (also known as PRKRA), andArgonaute (AGO)1–4 (also known as EIF2C1–4) mediate the processing of pre-miRNA and the assembly of the RISC
(RNA-induced silencing complex) in humans. One strand of the duplex remains on the Ago protein as the mature miRNA,
whereas the other strand is degraded. Ago is thought to be associated with Dicer in the dicing step as well as in the RISC
assembly step. In D. melanogaster, Dicer 1, Loquacious (LOQS; also known as R3D1) and AGO1 are responsible for thesame process. In flies, most miRNAs are loaded onto AGO1, whereas miRNAs from highly base-paired precursors are
sorted into AGO2. The figure shows the mammalian processing pathways with fly components in brackets. b | Canonicalintronic miRNAs are processed co-transcriptionally before splicing. The miRNA-containing introns are spliced more
slowly than the adjacent introns for unknown reasons. The splicing commitment complex is thought to tether the introns
while Drosha cleaves the miRNA hairpin. The pre-miRNA enters the miRNA pathway, whereas the rest of the transcript
undergoes pre-mRNA splicing and produces mature mRNA for protein synthesis. c | Non-canonical intronic small RNAs
are produced from spliced introns and debranching. Because such small RNAs (called mirtrons) can derive from small
introns that resemble pre-miRNAs, they bypass the Drosha-processing step. Some introns have tails at either the 5′ endor 3′ end, so they need to be trimmed before pre-miRNA export. m7G, 7-methylguanosine.

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REVIEWS
Because there are several different Ago proteins ina cell, small RNAs have multiple choices of Ago pro‑teins during RISC assembly. In D. melanogaster, themajor factor that determines small RNA sorting is
the structure of the precursor 93,94. miRNA duplexes
with central mismatches are preferentially sorted into
AGo1, whereas perfectly matching siRNA duplexes areincorporated into AGo2. A similar sorting mechanism
functions in C. elegans, in which the Ago‑like proteins
AlG‑1 and RDE‑1 associate with small RNAs frommismatched precursors and perfect dsRNA precursors,
respectively 95. The Dicer 2–R2D2 complex seems tofunction as a gatekeeper in D. melanogaster by favouring
perfectly matching siRNA duplexes and discriminating
against miRNA duplexes with mismatches, although
the molecular mechanism for this is not fully under‑stood. In humans, all four Ago proteins, AGo1–4, bindto miRNAs with only marginal differences in miRNArepertoire96–98, which suggests that human AGo1–4
might not have significantly differentiated functions.
The ends of miRNAs are often heterogeneous owing
to either the addition or deletion of 1–2 nt 98,99. Sequencevariations are found at both 5′ and 3′ ends, althoughthe 3′ ends tend to be much more variable than the
5′ ends. The mechanisms of the variations of the 5′ end
are unknown, but they might be explained by imprecise
or alternative processing by RNase III. Changes in the
5′ terminus result in shifts of the seed sequences (2–7 nt
from the 5′ end), which alter the target specificity of themiRNA. The 3′ end often contains untemplated nucleo‑
tides (mostly uracil and adenine), which must be added
after processing by unknown terminal uridyl/adenyltransferases. Deletions of the 5′‑ and 3′‑end nucleotides
are also often observed, which are probably due toexonucleolytic activities.

Regulation of miRNA biogenesis
Expression profiling studies indicate that most miRNAs
are under the control of developmental and/or tissue‑
specific signalling 100. Precise control of miRNA levels is
crucial to maintain normal cellular functions, and dys‑
regulation of miRNA is often associated with humandiseases, such as cancer 101.
Transcriptional control. Transcription is a major
point of regulation in miRNA biogenesis. Numerous
Pol II‑associated transcription factors are involved intranscriptional control of miRNA genes. for instance,
myogenic transcription factors, such as myogenin andmyoblast determination 1 (MyoD1), bind upstream of
miR‑1 and miR‑133 loci and induce the transcriptionof these miRNAs during myogenesis102–104. Some miRNAs
are under the control of tumour‑suppressive or onco‑
genic transcription factors. The tumour suppressor p53
activates the miR‑34 family of miRNAs (for a review, see
REF. 105), whereas the oncogenic protein MyC trans‑
activates or represses a number of miRNAs that are
involved in the cell cycle and apoptosis106,107. Epigenetic
control also contributes to miRNA gene regulation; themiR‑203 locus frequently undergoes DNA methylation in
T‑cell lymphoma but not in normal T lymphocytes108.

Post-transcriptional regulation. Drosha processing
confers another important point of regulation. miR‑21
is induced in response to bone morphogenetic protein(BMP)/transforming growth factor‑β (TGfβ) signallingwithout transcriptional activation109. It was proposedthat SMAD proteins activated by BMP/TGfβ interact
with Drosha and DDX5 (also known as p68) to stimulate
Drosha processing, although the detailed mechanism forthis remains unclear. Drosha processing of pri‑miR‑18
is dependent on the heterogeneous ribonucleoprotein
particle A1 (REFs 110,111). How many of such regulatory
factors exist is unclear, but it is plausible that nuclear RNA‑binding proteins influence miRNA processing throughspecific interactions with a subset of pri‑miRNAs.
The let‑7 miRNAs show interesting expression pat‑terns112. The primary transcript of let‑7 (pri‑let‑7) is exp‑
ressed in both undifferentiated and differentiated ES cells,
whereas mature let‑7 is detected only in differentiatedcells, indicating that let‑7a might be post‑transcription‑
ally controlled113–115. Similar post‑transcriptional inhibi‑tion of let‑7 also takes place in tumour cells114. Recent
studies show that an RNA‑binding protein, lIN28, is
responsible for the suppression of let‑7 biogenesis116–119.
Several different mechanisms of lIN28 action havebeen proposed: blockage of Drosha processing 116,117,
interference with Dicer processing 118,119 and terminal
uridylation of pre‑let‑7 (REF. 119). Given the cytoplasmic
localization of lIN28 (REF. 120) and its strong interaction
with pre‑let‑7 (but not with pri‑let‑7)119, lIN28 is likely
to function mainly in the cytoplasm by interfering withpre‑let‑7 processing and/or by inducing terminal uridyl‑
ation of pre‑let‑7. The u tail (~14 nt) that is added to the
3′ end of pre‑let‑7 blocks Dicer processing and facilitates
the decay of pre‑let‑7 (REF. 119). It is unknown how wide‑
spread this type of regulation is and which enzyme is
responsible for pre‑miRNA uridylation.
Turnover of miRNA is a largely unexplored area. RNA
decay enzymes might target not only mature miRNAs but
also the precursors (pri‑miRNAs and pre‑miRNAs).
once bound to Ago proteins, mature miRNAs seemto be more stable than average mRNAs; the half‑life of
most miRNAs is greater than 14 hours121. However, cer‑tain miRNAs (for example, miR‑29b) might be degradedmuch more rapidly than other miRNAs121, which suggests
a specific recognition of miRNA sequences by nucleases.
The 3′→5′ exonuclease ERI1 (also known as THEX1) was
previously shown to be responsible for the degradationof siRNAs in C. elegans 122. A group of exoribonucleases
named small RNA degrading nuclease (SDN) proteins
were recently reported to affect the stability of miRNAs inplants123. However, it remains unclear which nucleases are
responsible for miRNA degradation in animals.
RNA editing is another possible way of regulating
miRNA biogenesis. The alteration of adenines to inosines,
a process that is mediated by adenine deaminases(ADARs), has been observed in miR‑142 (REF. 124) andmiR‑151 (REF. 125). Because the modified pri‑miRNAs or
pre‑miRNAs become poor substrates of RNase III pro‑teins, editing of the precursor can interfere with miRNAprocessing. Editing can also change the target specificityof the miRNA if it occurs in miRNA sequences126.

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REVIEWS
An increasing number of miRNAs are controlled at
the post‑transcriptional level, yet the mechanisms ofregulation remain poorly understood for most miRNAs.
miR‑138 is specifically expressed in neuronal cells,
whereas its expression is suppressed at the Dicer‑mediatedprocessing step in non‑neuronal cells127. Human miR‑31,
miR‑128 and miR‑105, however, might be controlled at thenuclear export step because the precursors are retained in
the nucleus without producing mature miRNA in certain
cell types128. Mature miR‑7, miR‑143 and miR‑145 show
reduced expression in cancer cells compared with normal
tissue, although the precursor levels are similar between
the tumour and normal tissues, which suggests that post‑transcriptional misregulation occurs in cancer cells129,130.
Feedback circuits in miRNA networks. miRNA biogen‑esis is controlled by multiple layers of feedback loops
that involve the biogenesis factors, the miRNAs them‑selves and their targets131. Two types of feedback circuits
are frequently observed: single‑negative feedback anddouble‑negative feedback. Single‑negative feedback
usually results in stable or oscillatory expression of bothcomponents, whereas double‑negative feedback serves
as a bistable switch that results in mutually exclusive
expression.
levels of Drosha and Dicer are controlled by single‑
negative feedback to maintain the homeostasis of miRNA
production52,132,133. Drosha constitutes a regulatory circuit
together with DGCR8; Drosha downregulates DGCR8 bycleaving DGCR8 mRNA, whereas DGCR8 upregulates
Drosha through protein stabilization52,191. This loop seems
to be highly effective because even when the DGCR8 genecopy number is reduced by one‑half in Dgcr8 hetero‑
zygous cells, an almost normal level of DGCR8 protein is
produced and miRNA levels are also unaffected. Human
Dicer is controlled by its own product, let‑7, which binds
to the 3′ uTR and coding region of the Dicer mRNA132,133.
This might explain why Dicer knockdown is often moretransient and moderate than knockdown of other genes.
furthermore, numerous examples of single‑negative feed‑back have been described in worms, flies and mammals,
in which a transcription factor that transactivates miRNA
is itself repressed by that same miRNA131.
Double‑negative feedback control is also often used
as an effective genetic switch of specific miRNAs during
differentiation. one interesting example is the conserved
loop that involves let‑7 and lIN28 (REFs 116,117,119,134).
let‑7 suppresses lIN28 protein synthesis, whereas lIN28
blocks let‑7 maturation. The miR‑200 family and thetranscriptional repressors ZEB1 and ZEB2 also consti‑
tute a double‑negative feedback loop that functions inepithelial–mesenchymal transition135.

Retrotransposon
A transposon that mobilizes
through RNA intermediates;
the DNA elements aretranscribed into RNA and thenreverse-transcribed into DNA,
which is inserted at new sites
in the genome.

Piwi-interacting RNAs
piRNAs were originally discovered during small RNA
profiling studies of D. melanogaster development 136,137.
These studies uncovered a subset of endogenous, germ‑cell‑specific small RNAs (24–29 nt) that were clearly
distinct in their size from miRNAs. Most of this longer
species corresponded to intergenic repetitive elements,
including retrotransposons. Therefore, they were termed

repeat‑associated small interfering RNAs (rasiRNAs)137.
Earlier studies in D. melanogaster suggested that small
RNAs that correspond to retrotransposons might beinvolved in the silencing of transposable elements136. In
addition, PIWI protein was shown to be essential for self‑
renewal of germline stem cells138–140 and for transposon
mobility control141,142. Aubergine (AuB), another Piwi‑
subfamily protein, is required for pole‑cell formation143
and for repression of retrotransposons144. later, Vagin et al.
showed that piwi and aub mutations resulted in a loss ofrasiRNA accumulation in ovaries14, strengthening theconnection between rasiRNAs and Piwi proteins. The
physical interaction of rasiRNAs with Piwi proteins was
revealed by immunopurification of Piwi complexes,
mostly by using specific antibodies against each of
the Piwi proteins, including the third member of thesubfamily, AGo3 (REFs 14,145–148) (BOX 1; TABLE 1).
The Piwi‑subfamily proteins in mice (MIWI, MIlI andMIWI2; also known as PIWIl1, PIWIl2 and PIWIl4,
respectively)149–152, as well as those of zebrafish (Ziwi andZili; also known as Piwil1 and Piwil2, respectively)153,
were also shown to be associated with small RNAs that
resemble rasiRNAs. These small RNAs (24–31 nt) weretermed piRNAs. Mutations in Mili and Miwi2 derepressedtransposable elements, suggesting a function for mouse
piRNAs in transposon control154–156. on the basis of theircommon features, D. melanogaster rasiRNAs are now also
referred to as piRNAs155,157.
piRNA biogenesis in flies. piRNAs are produced fromintergenic repetitive elements on chromosomes. Most
piRNAs can be mapped to particular loci, which are
termed piRNA clusters. Interestingly, Piwi‑associatedpiRNAs include those that are derived from a part‑icular piRNA cluster locus, flamenco, on the X chromo‑
some146. The regulatory system that leads to such locus
discrimination remains unclear. furthermore, piRNAs
that are associated with each of the D. melanogaster Piwis
differ in size from each other 146,147. The differences insubcellular localization, the expression patterns andthe associated proteins might influence the selective
piRNA association (BOX 1; TABLE 1).
AuB‑ and PIWI‑associated piRNAs are mainly derived
from antisense transcripts of retrotransposons, whereas
AGo3‑associated piRNAs arise mostly from sense tran‑
scripts14,145–148. Nucleotide bias is also observed: AuB‑ andPIWI‑associated piRNAs show strong preferences for
uracil at their 5′ ends, whereas AGo3‑associated piRNAs
mostly have adenine at nucleotide 10 but show no bias
at the 5′ ends146,147. AuB‑associated piRNAs frequently
show complementarity to AGo3‑associated piRNAs in
their first 10 nt 146,147. Judging from the fact that piRNAs
accumulate in Dicer mutant ovaries14, piRNA productionin the fly ovary does not depend on Dicer. The require‑ment for Drosha has not been formally tested. So, how
are piRNAs produced without Dicer? one hypothesis isthat the Piwi proteins themselves might be involved inpiRNA biogenesis, given that they possess slicer nucle‑
ase activity; Piwi cleaves target RNA at between positions
10 and 11 relative to the 5′ end of the associated small
RNAs145. Based on this and the characteristics of piRNAs

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REVIEWS
piRNA loci(intergenic repetitive elements, active transposon genes and piRNA clusters)++––
Transcription

piRNA precursors

MIWI2
Nucleus?

?

?
Cytoplasm

Primary processing
Primary
processing

Loading
MIWI

MIWI2

MIWI2
MILI

Secondary
MIWI2 processing
MILI(ping-pong cycle)
MILI

MILI

MIWI
Loading

MILI

Figure 3 | pirNa biogenesis pathway. Piwi-interacting RNAs (piRNAs) of 24–31
nucleotides (nt) are processed from single-strandedNature
RNA precursors
that are transcribed
Reviews | Molecular
Cell Biology
from intergenic repetitive elements, transposons or large piRNA clusters. piRNAs
associate with Piwi-subfamily proteins; they are not dependent on either Drosha or
Dicer, although the requirement for Drosha has not been formally tested. They mightinstead use the nuclease activity of the Piwi proteins themselves for their processing.
piRNA biogenesis involves primary and secondary processing mechanisms. The strand
bias of piRNAs in secondary processing differs between mice and flies (for this reason,
fly components are not shown). Primary processing and loading might occur in thecytoplasm because Piwi proteins (MIWI and MILI; also known as PIWIL1 and PIWIL2,
respectively) are localized in the cytoplasm. Factors that are needed for primaryprocessing are unknown. In the secondary processing step (ping-pong cycle), MILI
introduces a cleavage in the precursor and thereby defines the 5′ end of piRNA, which issubsequently accepted by MIWI2 (also known as PIWIL4). MIWI2 also cleaves the
opposite strand precursor through its slicer activity, generating the 5′ end of the piRNAthat subsequently binds to MILI. The nuclease that creates the 3′ end of piRNA is
unknown. MIWI2 seems to be transported into the nucleus.

described above, a model for piRNA biogenesis has been
proposed146,147. AuB or PIWI that is associated with anti‑sense piRNA cleaves sense retrotransposon transcripts,
and this cleavage creates the 5′ ends of sense piRNA thatin turn associates with AGo3. Newly made AGo3 that is
associated with sense piRNA subsequently cleaves anti‑sense retrotransposon transcripts and thereby makes
the 5′ end of antisense piRNAs that subsequently bindto AuB or PIWI. factors that are necessary for the for‑mation of the 3′ end of piRNAs have yet to be identified.
These reciprocal reactions, known as the ping‑pong cycle,
would occur continuously in vivo, thereby amplifying thepiRNA population146,147. Concomitantly, retrotransposon
silencing can be maintained.

Mutations in several genes, such as those that encode
the putative nucleases Squash and Zucchini158, andothers such as Spindle‑E14, Krimper 159 and Maelstrom160,
cause the depletion of piRNAs in fly ovaries, suggestingthat they are involved in piRNA biogenesis. However, theprecise roles of these proteins remain elusive. piRNAs
derived from flamenco (flam‑piRNAs) are apparently
independent of the ping‑pong pathway because they arefound only in PIWI. This separate pathway for flam‑piRNAs is now referred to as the primary processing
pathway 161.
How does such a piRNA biogenesis cycle initiate
during development? In D. melanogaster, at least AuB
and possibly PIWI are deposited for the next generation
by germline transmission146,148. The maternal loading of
Piwi proteins into embryos was also observed in fish153.
A recent study shows that piRNAs that are maternallyinherited to embryos have an epigenetic regulatory rolein transposon silencing 162. This study explains how aphenomenon called hybrid dysgenesis is related to theloss of piRNA inheritance. Transmitted piRNAs, presum‑
ably in a form that is associated with Piwi proteins, most
likely act as primary ‘seeds’ that initiate an amplification
cycle of piRNA biogenesis in embryos, and thus, the cycle
could operate between generations.
piRNA biogenesis in mice. In mammals, two classesof Piwi‑interacting RNAs (piRNAs) have been identi‑
fied149–152,163. one class of mouse piRNAs (pre‑pachytene
piRNAs) is expressed before meiotic pachytene and isderived from repeat‑ and transposon‑rich clusters149.
In this regard, the pre‑pachytene piRNAs are similar to
D. melanogaster piRNAs. Pre‑pachytene piRNAs inter‑
act with two members of the mouse Piwi proteins, MIlIand MIWI2 (REFs 155,156,164). The other class of piRNAs
becomes abundant during the pachytene stage and asso‑
ciates with MIlI and MIWI150,155,164. These pachytene
piRNAs remain an enigma, as their sequences give noclues as to their possible targets. The pachytene piRNAs
are extremely abundant in spermatocytes: >80,000 dis‑tinct species are derived from large genomic clusters
of up to 200 kilobases. These piRNA clusters exhibit amarked strand asymmetry, as if the piRNAs are processedfrom one or a few huge transcripts. Intriguingly, piRNAsequences are not conserved among mammals. However,they are produced from syntenic regions of the mouse, rat
and human genomes.
Data from studies of mouse piRNAs support the ideathat the ping‑pong mechanism originally proposed inD. melanogaster might also apply to mouse pre‑pachytene
piRNAs164 (FIG. 3). However, the different compositionsof the embryonic, neonatal and adult piRNAs suggestthat the biogenesis cycle does not continue throughout
male germ‑cell development. In addition, pachytene
piRNAs do not show the obvious characteristics that are
observed for piRNAs produced by the ping‑pong path‑way. Thus, the primary processing pathway is likely to
be at work as in the case of D. melanogaster 164, although
it is currently unclear whether or not the primaryprocessing pathways in flies and mice are mechanicallysimilar (FIG. 3).

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REVIEWS
endo-siRNA loci(intergenic repetitive elements, pseudogenes and endo-siRNA clusters)
+
+
–

–

–

Transcription

Transcription

endo-siRNA precursors

cis-dsRNA

Hairpin-typetrans-dsRNA

Nucleus?
Cytoplasm
Exogenous dsRNA

Dicer

Dicer
TRBP or PACT?

TRBP or PACT?

AGO1–4 AGO1–4
exo-siRNA

AGO2

AGO2

endo-siRNA

Figure 4 | exo- and endo-sirNa biogenesis pathway. Exogenous small interfering RNAsNature
Reviews | double-stranded
Molecular Cell Biology(exo-siRNAs; see inset) are derived from experimentally
introduced
RNAs (dsRNAs) or viral RNAs. Human exo-siRNAs are loaded onto Argonaute (AGO)1–4(also known as EIF2C1–4), but only AGO2 has slicer activity. In flies, a complex thatcomprises Dicer 2 and R2D2 cleaves the exo-siRNA precursor, and AGO2 accept theRNAs. Endogenous siRNA (endo-siRNA) precursors are derived from repetitive sequences,sense–antisense pairs or long stem-loop structures. In humans, Dicer and AGO2 functionin the endo-siRNA pathway; it has not been determined whether human endo-siRNAs
associate with other Ago proteins, or whether TRBP (TAR RNA-binding protein; also
known as TARBP2) and/or PACT (also known as PRKRA) are involved in this pathway (seequestion mark). In flies, Dicer 2, together with Loquacious (LOQS; also known as R3D1),
processes the precursor into ~21-nt endo-siRNAs, which are accepted by AGO2.

RNA-dependent RNA
polymerase
An RNA polymerase thattranscribes RNAs from RNA
templates.

As in flies, some mouse piRNAs correspond to trans‑poson sequences, implying that they also function insilencing selfish DNA elements. However, even though thefunctional outcome is similar, whether they use the same
strategy for silencing is questionable. In mouse testes,
the promoter regions of particular retrotransposons are
methylated. Mutations in Mili and Miwi2 eliminatedDNA methylation of long interspersed nuclear element 1(lINE1) and intracisternal A particle (IAP) and led to maleinfertility 154,155. MIlI and MIWI2 have essential roles inestablishing de novo DNA methylation of transposons
in fetal male germ cells156,164. These results suggest thatmouse piRNAs are involved in transcriptional silencingof target genes through DNA methylation.
unlike animal miRNAs but similar to plant miRNAs,
piRNAs have a 2′‑O‑methyl modification at their 3′
ends14,165–168. This modification is carried out by homo‑logues of A. thaliana HEN1 methyltransferase169 (known
as HEN1 or PIMET in flies)165,166. It was also shown that
HEN1 associates with Piwi proteins in ovaries. However,the biological significance of the 2′‑O‑methyl modifica‑tion remains unknown because D. melanogaster mutantsof HEN1 show no obvious malfunctions165.

Endogenous siRNAs
Deep sequencing of small RNAs in D. melanogastersomatic tissue, cultured cells and ovaries has identified
a novel class of ~21‑nt RNAs17–20,170–172. These RNAs arederived from transposon transcripts, sense–antisensetranscript pairs and long stem‑loop structures17–20,170–172.
Specific association of these endo‑siRNAs with AGo2 wasshown biochemically in flies19,20. Extensive small‑RNA
profiling studies in mice also revealed that numerous
types of endo‑siRNAs are present in oocytes15,16 and lessabundantly in ES cells17. Mouse endo‑siRNAs are also
dependent on the Ago‑family protein (mouse AGo2;
also known as EIf2C2)15.
Apart from the endo‑siRNA pathway, D. mela‑nogaster AGo2 functions in the RNA interference path‑way by associating with exogenous siRNAs (exo‑siRNAs;
which are ~21 nt in length) that arise from ectopicallyintroduced long dsRNAs or siRNA duplexes96. forexample, flies use the RNA interference mechanism
to defend against viruses that produce dsRNAs during
infection173,174.
Numerous endogenous siRNAs have been describedin other species, such as plants and C. elegans 29. Endo‑siRNA pathways in plants and worms are more complex
than those in flies and mammals, and they possess RNAdependent RNA polymerases (RdRPs)29 for the generationof endo‑siRNAs. Because flies and mammals lack RdRP,
fly and mammalian endo‑siRNAs should be categorized
as a new group of endo‑siRNAs that are produced in an
RdRP‑independent manner.
Endo-siRNA processing by Dicer 2 in flies. The size dis‑tribution of endo‑siRNAs is narrow and concise (~21 nt)
compared with, for example, the 21–23‑nt lengths ofmiRNAs. The biogenesis system probably holds the key
to this difference. Similar to exo‑siRNAs, but different
from miRNAs, the processing of endo‑siRNAs is depend‑ent on Dicer 2 rather than Dicer 1 (REFs 18–20,170–172).
Interestingly, unlike exo‑siRNAs, which require R2D2
(REF. 88), endo‑siRNAs, particulary those that originate
from long stem‑loops, rely on loQS, which functionsin the miRNA pathway 18–20,73,75,170–172. Dicer 2 seems to beable to associate with loQS in D. melanogaster cells146.
How and why loQS selects Dicer 2 over Dicer 1 as apartner in endo‑siRNA processing is unknown. It would
also be interesting to understand how the Dicer 2–loQS
complex recognizes its substrates and determines thecleavage sites.
Endo‑siRNA precursors are mainly produced fromsense–antisense pairs derived from transposons. They
can also arise from convergent transcription of pro‑tein‑coding genes and from unannotated regions of the
genome19,20,170,171. These transcripts are not necessarilytranscribed from the same loci; thus, the dsRNA pre‑cursors tend to have natural mismatches and bulges.
The second type of endo‑siRNA precursor is a single‑
stranded, but self‑hybridizing, transcript that forms
a long stem‑loop structure19,20,170,171. They are distin‑
guished from miRNA precursors by the extended lengthof the stems. Interestingly, a significant number ofendo‑siRNAs (~20% of AGo2‑associated endo‑siRNAs

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REVIEWSin S2 cells) show sequence substitutions19. As most of
the mutations found were A–G substitutions, this isprobably due to RNA editing by ADAR175. The editing
activity of ADAR is restricted in the nucleus and accepts
only dsRNAs as the substrate. Therefore, the dsRNA
precursors must have been formed in the nucleus, inwhich they serve as the substrates for ADAR. This post‑transcriptional nucleotide modification causes further
tiny bulges in the precursors. These two structuralfeatures of endo‑siRNA precursors (long dsRNA struc‑tures and bulges) might be recognized selectively by theprocessing factors Dicer 2 and loQS. Dicer 2 might berequired for long dsRNA processing and for interaction
with AGo2. loQS might contribute to the binding ofDicer 2 to mismatched dsRNAs and to their assembly
into RISC.
Some piRNA‑generating loci can be a source of endo‑
siRNAs19, although Czech et al.20 reported a controversial
observation that only a few piRNA loci produce endo‑
siRNAs. like piRNAs, endo‑siRNAs have 2′‑O‑methylgroups at their 3′ ends in flies, which might be added by
the methyltransferase HEN1 (REF. 19). However, piRNAs
and endo‑siRNAs are clearly different with regard totheir size (24–29‑nt compared with 21‑nt), their protein
partners (Piwi proteins compared with AGo2) and thecells in which they are expressed (mostly germ cells com‑pared with ubiquitous expression). Their editing status isalso unequal; endo‑siRNAs can be edited by ADAR, but
this is unlikely to be the case for piRNAs, which implies
that endo‑siRNAs are generated from dsRNAs, whereas
piRNAs originate from ssRNAs. Retrotransposons canbe transcribed in both directions. However, the amountsof the sense and antisense products may not be equal.
This would result in a mixture of dsRNAs and ssRNAs.
Regardless of editing, the dsRNAs are transported tothe cytoplasm by an unknown mechanism, and serve as
substrates for Dicer 2 activity to generate endo‑siRNAs.
The single‑stranded transcripts are also transported tothe cytoplasm, probably through the mRNA export path‑way, and become substrates for the piRNA‑producing
machineries.
Endo-siRNA biogenesis in mouse oocytes. like D. melano‑
gaster endo‑siRNAs, mouse endo‑siRNAs are ~21 nt
long and are derived from a range of sources, includingtransposable elements15,16. Common features can also befound in their biogenesis. Dicer is required for mouseendo‑siRNA production, as is Dicer 2 in D. melanogaster(FIG. 4). The precursors of mouse endo‑siRNAs are tran‑
scripts that contain long hairpin structures or dsRNAs
that are derived from sense–antisense pairs. Some ofthese sense–antisense pairs (cis‑endo‑siRNA precur‑sors) are transcribed from convergent transcription of
the same loci, whereas others (trans‑endo‑siRNA precur‑sors) are derived from two similar but separate loci. It was
shown that pseudogene transcripts can generate siRNAs
by annealing to their cognate functional transcripts, andthat the functional genes are upregulated in the Dicer andAgo2 mutants15,16. Thus, pseudogenes might have a regu‑
latory role by suppressing their functional counterparts
through an RNA silencing mechanism.

Interestingly, mouse oocytes produce both endo‑
siRNAs and piRNAs, whereas mouse testes express only
piRNAs15,16. Thus, in ovaries, transposon silencing isensured by two pathways: piRNA and endo‑siRNA path‑ways. During evolution, transposons in male and female
mouse germ lines may have been regulated in differ‑ent ways. Although some of them still remain active infemales (in which they might confer some advantage
in oocyte development), the endo‑siRNA pathway might
have been lost in males.

conclusions
Tens of thousands of small RNAs have been discovered,and more small RNAs are expected to be discovered
owing to rapidly expanding sequencing power. This isan exciting era in RNA biology, but at the same time weare facing multiple challenges. Most of all, it is difficult todistinguish between functional small RNAs and non‑functional ‘noises’. In fact, there are numerous misanno‑tated small RNA entries in databases, which are apparently
degradation products from other longer RNA species.
Evolutionary conservation confers compelling evidence
for the functionality of the cloned RNAs, but the small
RNAs will eventually need to be experimentally vali‑
dated to prove their functionality. Confirmation of theirexpression and their dependence on the Ago proteins
will first be needed to validate individual small RNAs.
Dependence on other biogenesis factors, such as Drosha,
DGCR8 and Dicer, will also be useful in validatingsmall RNAs as well as in classifying them17.
The miRNA biogenesis pathway is well studied incomparison to piRNA and endo‑siRNA pathways,
although many questions remain unanswered. A moredetailed understanding of the mechanism awaits the
structures of the complexes, including Microprocessor,
EXP5 and Dicer–RISC in association with the substrate
RNAs. Many factors are implicated in miRNA biogen‑esis, such as DDX5, DDX17 (also known as p72), NfAR
(also known as Ilf3)40,176,177, SNIP1 (REF. 178), PACT78
and Mei‑P26 (REF. 179), the biochemical roles of which
are unknown. furthermore, additional protein factors in
the pathway need to be identified. for instance, we do
not know the identities of the factors that are involved inmiRNA turnover in animals. We also need to understandthe significance and enzymology of the modificationsof small RNAs, such as uridylation and adenylation of
miRNAs. Moreover, we have yet to learn about the aux‑
iliary factors that regulate miRNA maturation. It would
be interesting to understand how miRNA biogenesis iscontrolled by these factors in response to various cellularsignals. Developmental and environmental cues should beinterpreted accurately by these factors to correctly operate
the miRNA network.
The piRNA pathway is still largely elusive. piRNAs
are highly diverse in sequence, and many of them arederived from non‑repetitive sequences, especially inmice. Because the ping‑pong model does not explain all
characteristics of piRNAs, it is likely that an additional
biogenesis pathway exists for certain piRNAs, particularly
those that are associated with Piwi in D. melanogaster andpachytene piRNAs in mice. It would also be interesting to136 | fEBRuARy 2009 | VoluME 10

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REVIEWS
understand the mechanistic details of how piRNAs relate
to heterochromatin formation. Identification and analysesof the proteins associated with the Piwi proteins mightprovide clues.
There are numerous other small RNAs, like mirtrons,that are generated through non‑canonical pathways.
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pathways remain poorly understood. Although most ofthese non‑canonical small RNAs are less abundant andless conserved than major small RNAs, such as miRNAs,
they might have species‑specific functions that are not yet
fully appreciated.
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including transcription, pre‑mRNA splicing and mRNA
decay. Given that intronic miRNAs are processed co‑transcriptionally, it is tempting to speculate that theseprocesses (transcription, splicing and Drosha processing)

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Acknowledgements

We are grateful to H. Siomi, T. Watanabe, Y. Watanabe andS. Kuramochi-Miyagawa for helpful discussions and comments.
This work was supported by the Creative Research Initiatives
Program (V.N.K.), the BK21 Research Funds from the Ministryof Education (J.H.) from Science and Technology of Korea, New
Energy and Industrial Technology Development Organization(NEDO) grants (M.C.S.) and Core Research for Evolutionary
Science and Technology (CREST) from the Japan Science andTechnology Agency (JST) (M.C.S.). M.C.S. is Associate
Professor of Global COE for Human Metabolomics Systems
Biology by the Ministry of Education, Culture, Science andTechnology, Japan.

DAtABAsEs
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=geneflamenco
The miRNA Registry: http://microrna.sanger.ac.uk/
sequenceslet-7 | lin-4 | miR-1 | miR-7 | miR-29b | miR-31 | miR-34 |miR-105 | miR-128 | miR-133 | miR-138 | miR-142 | miR-143 |miR-145 | miR-151 | miR-200 | miR-203 | pri-miR-18
UniProtKB: http://www.uniprot.org
AGO1 | AGO2 | AGO3 | AGO4 | ALG-1 | AUB | DGCR8 | Dicer 1| Dicer 2 | Drosha | ERI1 | EXP5 | LIN28 | LOQS | MILI | MIWI |MIWI2 | MYC | MYOD1 | myogenin | p53 | PACT | PIWI | R2D2 |RDE-1 | TRBP | XRN2 | ZEB1 | ZEB2 | Zili | Ziwi

FURtHER iNFoRMAtioN
V. Narry Kim’s homepage: http://www.narrykim.org
Mikiko C. Siomi’s homepage: http://web.sc.itc.keio.ac.jp/
dmb/sindex.html
miRNA database: http://microrna.sanger.ac.uk
all liNkS are active iN the oNliNe pdf

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