REVIEWS

Diversifying microRNA sequence

and function
Stefan L. Ameres1 and Phillip D. Zamore2
Abstract | MicroRNAs (miRNAs) regulate the expression of most genes in animals, but we are
only now beginning to understand how they are generated, assembled into functional
complexes and destroyed. Various mechanisms have now been identified that regulate
miRNA stability and that diversify miRNA sequences to create distinct isoforms.
The production of different isoforms of individual miRNAs in specific cells and tissues may
have broader implications for miRNA-mediated gene expression control. Rigorously testing
the many discrepant models for how miRNAs function using quantitative biochemical
measurements made in vivo and in vitro remains a major challenge for the future.

Argonaute
(AGO). Proteins that are guided
to mRNA targets by small
silencing RNAs. AGO proteins
can also serve as scaffolds to
bind secondary silencing
factors such as the GW‑repeat-
containing protein GW182.
Primary miRNAs
(pri-miRNAs). Polyadenylated,
7‑methylguanosine-capped
RNA polymerase II transcripts
containing a stem–loop
structure that serves as a
substrate for the RNase III
enzyme Drosha in animals and
Dicer-like 1 (DCL1) in plants.
They are processed to liberate
a precursor miRNA and two
unstable, single-stranded
by‑products.
1
Institute of Molecular
Biotechnology of the Austrian
Academy of Sciences (IMBA),
Dr. Bohr-Gasse 3, 1030
Vienna, Austria.
2
Howard Hughes Medical
Institute and University of
Massachusetts Medical
School, 55 Lake Avenue
North, Worcester,
Massachusetts 01605, USA.
e‑mails: stefan.ameres@
imba.oeaw.ac.at; phillip.
zamore@umassmed.edu
doi:10.1038/nrm3611
Published online 26 June 2013
For more than a decade, miRNAs have enthralled the
biological community. Almost everything about micro-
RNAs (miRNAs) is small. At ~22 nucleotides long, they
are among the shortest functional eukaryotic RNAs. They
were discovered in a diminutive organism, Caenorhabditis
elegans, and their physiological relevance in regulating
plant and animal gene expression was underappreciated
for nearly a decade1,2. Finally, miRNA­s repress most of
the genes they regulate by just a tiny amount. Yet, col-
lectively miRNAs affect nearly all cellular pathways, from
developmen­t to oncogenesis.
The primary repository for miRNA sequences and
annotations, miRBase, debuted with just 218 miRNA loci3.
Since then, the application of high-throughput sequencing
to miRNA discovery in >193 different plant and anima­l
species has swelled that number to >21,264 loci that
produce >25,141 mature miRNAs. The human genome
alone comprises >1,500 hairpin structures that produce
detectable small RNAs, although the authenticity and
functional importance of many of these miRNA candi-
dates remain to be established4. Selective pressure during
evolution seems to have maintained the pairing between
miRNAs and more than half of all human protein-coding
genes5, suggesting that these tiny riboregulator­s control
the expression of most human proteins6.
The absence of miRNAs in fungi and the distinct
sequences, precursor structures and mechanisms of pro-
duction for plant and animal miRNAs suggest that the
miRNA pathway evolved at least twice7. Small silencing
RNAs, including miRNAs, are present in the earlies­t
diverging extant lineage of animal life, the sponge
Amphimedon queenslandica, suggesting that the emer-
gence of miRNAs herald the dawn of multicellular
animals8. In the plant kingdom, the miRNA pathway
presumably evolved before multicellularity, because the
unicellular alga Chlamydomonas reinhardtii produces
miRNAs that are similar to those of higher plants9.
For several model animals and plants, as well as
humans, we now know the majority of miRNAs and these
are inferred to be functional on the basis of their evolu-
tionary conservation. But we are only beginning to under-
stand the divers mechanisms that contribute to miRNA
production and assemble them into complexes with pro-
teins. For example, although it is established that miRNAs
direct Argonaute (AGO) proteins to repress mRNAs, the
mechanism of repression remains intensely debated.
Here, we review the mechanisms that diversify miRNA
sequence and function. We briefly outline the pathways
that generate miRNAs and regulate their production,
activity and longevity. We describe the mechanisms by
which miRNA sequences and functions can be altered, as
well as the processes that destroy rogue and old miRNA­s.
Finally, we consider the evidence for different models
of how miRNAs function and present a quantitativ­e
framewo­rk for evaluating these models.
Making miRNAs
Most miRNAs derive from longer, intramolecularly
double-stranded RNAs (dsRNAs; termed primary miRNA­s
(pri-miRNAs)) that are sequentially cleaved into shorter
intermediates by specialized ribonuclease III (RNase III)
enzymes that partner with specific dsRNA‑binding
protein­s 10,11 (FIG.  1) . Both transcriptional and post-
transcriptiona­l mechanisms regulate miRNA biogenesis
(for a detailed discussion, see REFS 12,13). A few miRNA­s
are produced by alternative pathways that replace

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REVIEWS

Standard pathway Alternative pathway standard biogenesis steps with RNA processing control
RNA Pol II from pre-mRNA splicing or RNA degradation pathways
m7Gppp
(for further details, see REF. 14).

miRNAs are transcribed by RNA polymerase II. RNA

mRNA splicing poly­merase II (Pol II) produces long pri-miRNAs from
independent genomic transcription units or from the
introns of protein-coding genes15–22. For miRNAs that
Drosha
arise from introns, splicing is not required for their pro-
Pri-miRNA
AAAA(n) Branched Mirtron
duction23, and the co‑transcriptional processing of pri-
m7Gppp miRNA into precursor miRNAs (pre-miRNAs) does not
Pasha or
Pri-miRNA DGCR8 Lariat debranching affect the splicing of the host pre-mRNA24,25.
Nucleus cropping
Drosha crops pri-miRNAs into shorter pre-miRNAs.
In animals, the RNase III enzyme Drosha converts pri-
Cytoplasm miRNA­s into pre-miRNAs, which are ~60 nucleotide
Exportin 5
stem–loop structures. Drosha excises one or more pre-
miRNAs from the pri-miRNA26–29. Thus, pri-miRNAs can
produce several pre-miRNAs. Polycistronic miRNAs
Pre-miR-451
Loqs or allow a single promoter to drive co‑expression of multi-
TRBP ple miRNAs and, in some cases, mediate the coordinated
Pre-miRNA Dicer expression of target factors in a single pathway 10.
Drosha acts as a component of a larger complex, the
microprocessor. This nuclear complex seems to exist
in at least two forms, a ~600 kDa complex of unknown
H
O
2′

miRNA–miRNA* function and a smaller heterodimer comprising Drosha

AGO2
duplex and its dsRNA-binding protein, named DGCR8 in mam-
mals and Pasha in other animals28–31. The collaboration
H
O
2′

Pre-miRNA of Pasha with Drosha defines a general paradigm in

HSC70 and HSP90 cleavage and
maturation
AGO
Pre-RISC
miRNA*
Mature RISC RISC
m7Gppp
(n)AAAA ORF
Figure 1 | MicroRNA biogenesis.  In the standard microRNA (miRNA) biogenesis
Nature Reviews | Molecular Cell Biology
pathway, primary miRNA (pri-miRNA) transcripts are processed by Drosha in the
nucleus and by Dicer in the cytoplasm. The pri-miRNA, which is transcribed by RNA
polymerase II (Pol II), begins with a 7‑methylguanosine cap (m7Gppp) and ends with a
3ʹ poly(A) tail. The pri-miRNA contains a stem–loop structure that is cleaved in the
nucleus by the endonuclease Drosha together with its double-stranded RNA
(dsRNA)-binding protein parner DGCR8 (in mammals) or Pasha (in flies). The resulting
precursor miRNA (pre-miRNA) is exported from the nucleus by exportin 5 and then
further cleaved by the endonuclease Dicer together with its dsRNA-binding partner
TRBP (transactivation-response RNA-binding protein; in mammals) or Loquacious
(Loqs; in flies) to liberate a miRNA–miRNA* duplex. Supported by the HSC70–HSP90
chaperone machinery, this duplex is loaded into an Argonaute (AGO) protein as a
dsRNA. Subsequent maturation steps expel the miRNA*, producing a mature
RNA-induced silencing complex (RISC). Alternative pathways (shown on the right)
typically replace individual steps of miRNA precursor processing. Pri-miRNA cropping
can be replaced with nucleases from other cellular pathways, including the general
RNA degradation machinery or pre-mRNA splicing factors. In such instances, the
pri-miRNA is generated from a branched mirtron structure that undergoes lariat
debranching. In the specific case of pre-miR‑451, the pre-miRNA escapes Dicer
processing after nuclear export and is instead directly loaded into the AGO2 protein,
which triggers its maturation into a single-stranded miRNA. 2ʹ OH, 2ʹ hydroxyl group;
HSP, heat shock protein; ORF, open reading frame.
small RNA biogenesis: dsRNA-binding proteins part-
ner with RNase III enzymes to restrict the substrates
they process, increase their affinity for a substrate or
improve cleavage site accuracy. The subcellular distribu-
tion of miRNA-producing enzymes and their substrates
implies that animal pri-miRNA­s are cropped in the
nucleus, whereas pre-miRNAs are processed in the cyto-
plasm26,32–36. The nuclear transport receptor exporti­n 5
(known as Ranbp21 (accession number CG12234)
in flies) recognizes the ends and the stem of the pre-
miRNA37 and exports the pre-miRNA from the nucleus
to the cytoplasm via the nuclear pore38–41.
Dicing makes miRNA–miRNA* duplexes. In the cytoplas­m,
a second RNase III enzyme, Dicer, liberates a ~22 nucleo-
tide miRNA–miRNA* duplex from the pre-miRNA42–45.
In flies, Dicer‑1 cleaves pre-miRNAs, whereas Dicer‑2
generates siRNAs46. Like Drosha, Dicer‑1 recognizes
defined RNA structures and then cleaves at a fixed dis-
tance away from the base of the pre-miRNA stem, cutting
off the loop to produce ~22 nucleotide mature miRNA–
miRNA* duplexes47,48. Like cropping of pri‑miRNA­s,
‘dicin­g’ of pre-miRNAs can also be bypassed. For exam-
ple, the hairpin of pre-miR-451 in zebrafish and mice is
too short to be recognized by Dicer; instead, it is directly
loaded into the RNA-induced silencing complex (RISC) for
further processing by AGO2 into a mature miRNA49–51.
Partner proteins shape Dicer function. Continuing the
theme of RNase III enzymes that partner with dsRNA-
binding proteins, Drosophila melanogaster Dicer‑1 part-
ners with two isoforms of the dsRNA-binding protein

476 | AUGUST 2013 | VOLUME 14 www.nature.com/reviews/molcellbio

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 REVIEWS

Flies and mammals Flies Plants

miRNA siRNA miRNA

– Hen1 + Hen1 – HEN1 + HEN1
and siRNA

HESO1 HEN1
AGO1 AGO2 AGO2
UUUUU 2′O-CH3
2′O-CH3
Highly complementary Tailing HEN1
target mRNA miRNA* miRNA*
Hen1
2′ OH 2′ O-CH3
NNN ? ? 3′-to-5′
exonuclease
Tailing 3′-to-5′exonucleolytic
trimming AGO
2′O-CH3 ? 2′O-CH3
Small RNA turnover siRNA stabilization Small RNA turnover Small RNA stabilization
Figure 2 | Small RNA turnover by tailing and trimming.  In the tailing-and-trimming pathway, the presence of a highly
complementary target RNA triggers both tailing and trimming of microRNAs (miRNAs),Natureultimately
Reviews | decreasing theirBiology
Molecular Cell
abundance in flies and mammals (left panel). The identity of the exonuclease is not known (denoted by the question
mark in the figure). In flies, Argonaute 2 (AGO2)‑bound siRNAs are protected from tailing and trimming because the
methyltransferase Hen1 (Hua enhancer 1) methylates siRNAs at the 2ʹ position of the 3ʹ terminal nucleotide. In the
absence of Hen1, siRNAs are subjected to target RNA-directed tailing and trimming (middle panel). Unlike fly Hen1,
plant HEN1 methylates both siRNA and miRNA duplexes before they are loaded into AGO. As in flies, loss of HEN1 in
plants prompts small RNA tailing, which is mediated by the plant terminal nucleotidyltransferase (TNTase) HEN1
SUPPRESSOR 1 (HESO1), ultimately resulting in 3ʹ-to‑5ʹ exonucleolytic decay (right panel). The normal triggers for
small RNA decay in plants are unknown.

Ribonuclease III
(RNAse III). Double-stranded
RNA-specific endoribonuclease
that generates products with
two nucleotide 3ʹ overhangs, a
5ʹ phosphate and a 3ʹ hydroxyl
group.
dsRNA-binding protein
Proteins containing
double-stranded RNA-binding
domains, which are ~70
amino acid motifs that bind
to RNA helices via their
2ʹ hydroxyl group and
phosphate backbone.
Precursor miRNAs
(pre-miRNAs). Stem–loop RNAs
comprising a single-stranded
loop that connects two partially
complementary sequences.
These sequences pair to form a
predominantly double-stranded
stem. Pre-miRNAs typically
have a 5ʹ phosphate and a
two nucleotide 3ʹ overhang,
allowing them to serve as
substrates for Dicer.
Drosha
The nuclear RNase III
endonuclease in animals that
cleaves the base of a stem–loop
structure contained in primary
microRNAs (pri-miRNAs) to
produce a precursor miRNA.
Collaborates with mammalian
DGCR8 (or Pasha in other
animals), which is its
double-stranded RNA-binding
protein partner.
Loquacious (Loqs), termed Loqs‑PA and Loqs‑PB.
These are required for efficient miRNA processing in
flies because they increase the affinity of Dicer‑1 for pre-
miRNAs52–55. Conversely, the Dicer‑2 partner protein
R2D2 prevents Dicer‑2 from processing pre-miRNAs,
restricting it to long dsRNA substrates56.
Alternative partner proteins refine the substrate spec-
ificity of individual Dicer proteins, and this may explain
how organisms such as worms and mammals produce
miRNAs and siRNAs with only a single enzyme.
Consistent with this, mammals have two Dicer
partners, transactivation-response RNA-binding pro-
tein (TRBP) and protein kinase R‑activating protein
(PACT)57–59.
In addition to their role in defining Dicer substrate
specificity, Dicer partner proteins can alter the position
of Dicer-mediated cleavage within a pre-miRNA55,60. For
example, TRBP, but not PACT, changes the site at which
mammalian Dicer cleaves a few pre-miRNAs, such as
pre-miR‑132. In flies, Loqs‑PB changes the length and
seed sequence of the miRNAs generated by Dicer‑1 from
a small subset of pre-miRNAs.
A seed for target recognition. After the sequential pro-
cessing of miRNA precursors, one of the two strands
of the miRNA duplex guides AGO proteins to comple-
mentary mRNA sequences to repress their expression
(see REFS 61,62 for detailed reviews of AGO loading). The
major determinant for AGO binding to its target mRNA is
a 6–8 nucleotide domain at the 5ʹ end of the miRNA.
AGO associates with this region to create the ‘seed’ (for
details, see REF. 63). Sequences that are complementary
to the seed (‘seed matches’) suffice to trigger a modest
but detectable decrease in the expression of an mRNA.
Seed matches can occur in any region of an mRNA but
are more likely to decrease mRNA expression when they are
in the 3ʹ untranslated region (3ʹ UTR)64–67. Because the
region used to create the seed is so short, more than half
of all protein-coding genes in mammals are regulated by
miRNAs, and thousands of other mRNAs seem to have
experienced negative selection to avoid seed matches with
miRNAs that are present in the same cells5,68–70.
Target RNAs alter miRNA stability
Binding of the AGO–miRNA complex to an mRNA
not only regulates gene expression (discussed in detail
below) but also alters the stability of the miRNA itself. For
example, binding of complementary target RNAs in flies
and mammals can direct miRNA destruction. miRNA
decay elicited by target binding was first reported for
antagomirs, which are chemically modified, synthetic
oligonucleotides used to inhibit miRNA function in cells
and in vivo71. We now know that antagomirs, as well as
highly expressed RNAs containing sites with extensive
complementarity to a miRNA, trigger miRNA degra-
dation (FIG. 2). This pathway involves both the addition
of adenosine or uracil to the miRNA (‘tailing’) and the
3ʹ-to‑5ʹ exonucleolytic resection of the miRNA 3ʹ end
(‘trimming’)72–74. Target RNA-directed tailing and trim-
ming of miRNAs require greater complementarity than
is typically found between miRNAs and their targets72,74.
This may be because the structural rearrangements
associated with target binding provide the tailing and
trimming enzymes access to the miRNA 3ʹ end, which
would otherwise reside in the AGO PAZ domain75–79.
Alternatively, all miRNAs may be susceptible to tailing

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REVIEWS
Dicer
An RNase III endonuclease that
is predominantly found in the
cytoplasm of animal cells and
the nucleus of plant and some
fungal cells. Dicer proteins
liberate mature microRNA–
microRNA* duplexes from
pre-microRNAs and siRNA
duplexes from long
double-stranded RNAs.
siRNAs
The ~21 nucleotide small
RNAs that mediate RNA
interference in plants, animals
and some fungi. Typically
produced by Dicer processing
of long double-stranded RNA
(dsRNA) precursors encoded in
the genome (endo-siRNAs) or
from exogenous dsRNA
sources (exo-siRNAs) such
as viruses.
RNA-induced silencing
complex
(RISC). A ribonucleoprotein
complex that consists of a
small RNA guide strand bound
to an Argonaute protein.
RISC mediates all RNA
silencing pathways, and it can
also include auxiliary proteins
that extend or modify its
function.
Seed sequence
A nucleotide motif in the
5ʹ domain of all small silencing
RNAs, which is organized by
Argonaute to determine
target-RNA recognition.
PIWI-interacting RNAs
(piRNAs). Small silencing RNAs,
25–35 nucleotides long, that
bind PIWI clade Argonaute
proteins in animals and silence
germline transposons. They are
thought to derive from
single-stranded RNA precursors
and do not require RNase III
enzymes for their maturation.
Exoribonuclease
Enzymes that successively
remove nucleotides from either
the 3ʹ (3ʹ-to‑5ʹ exoribonuclease)
or the 5ʹ end (5ʹ-to‑3ʹ
exoribonuclease) of RNA.
They catalyse phosphodiester
bond cleavage using water
(releasing nucleotide
monophosphates) or inorganic
phosphate (releasing
nucleotide diphosphates)
as a nucleophile.
Isomirs
microRNA variants containing
sequences that deviate from
miRBase-annotated or most
frequently observed species.
478 | AUGUST 2013 | VOLUME 14 www.nature.com/reviews/molcellbio
and trimming when bound to target RNAs, but the
slower dissociation of miRNAs from high-affinity sites
makes highly complementary RNAs more efficient at
triggering modification and degradation of miRNAs.
The endogenous functions of target RNA-directed
tailing and trimming of miRNAs in animals remain
elusive, and the proteins that mediate this pathway
are unknown. However, tailing and trimming can be
triggered by transcribed miRNA inhibitors, and use
of these inhibitors in adult mice phenocopies miRNA
loss‑of‑function mutations, suggesting that this pathway
is functionally relevant for miRNA-mediated regulation
of gene expression in vivo74,80,81. Moreover, some viruses
use a similar strategy to interfere with host miRNA
function during infection (for details, see REFS 82,83).
To what extent target RNA-directed miRNA degradation
contributes to the regulation of miRNA function in vivo
remains to be determined. But it is tempting to speculate
that the tailing-and-trimming pathway has contributed
to the evolution of target sites in mRNAs in animals, as
mRNAs rarely possess sufficient complementarity to
miRNAs to trigger miRNA destruction68,69.
In D. melanogaster, target RNA-directed small RNA
decay is restricted to the miRNA pathway. siRNA­s in
flies and PIWI-interacting RNAs (piRNAs) in all ani-
mals are protected from tailing and trimming by
2ʹ-O‑methylation of their 3ʹ ends, a modification that
is introduced during AGO loading by the methyltrans-
ferase HEN1 (Hua enhancer 1)72,84–86. Flies use these
protected siRNAs to target AGO2–RISC complexes for
the repression of extensively complementary targets.
In the absence of Hen1, these targets promote the tail-
ing, trimming and depletion of complementary endo­
genous siRNA­s86. HEN1 was first discovered in plants, in
which it methylates both miRNAs and siRNAs87,88. Loss
of HEN1 in plants provokes uridylation and 3ʹ-to‑5ʹ exo-
nucleolytic decay of miRNAs and siRNAs88–91. Plant and
animal HEN1 enzymes catalyse the same chemical reac-
tion92, but plant small RNAs are methylated as duplexes
before their loading into AGO88,92,93, whereas animals
methylate only a subset of single-stranded small RNAs,
typically piRNAs. In flies, and presumably in arthropods
more generally, HEN1 methylates siRNAs as the last step
in AGO2–RISC assembly 85, but mammalian siRNAs are
thought not to be HEN1 substrates.
Tailing and trimming distinguish endogenous from for-
eign small silencing RNAs. In flies, highly complemen-
tary target RNAs have not been found for miRNA­s bound
to AGO1 (the miRNA-binding AGO protein in flies).
Animal miRNAs generally lack a protectiv­e 2ʹ-O‑methyl
modification (FIG. 2). Introduction of RNAs with exten-
sive complementarity to one of these miRNAs triggers
tailing and trimming of the miRNA. Thus, in flies, Hen1
and the tailing-and-trimming pathway might collabo-
rate to distinguish self from non-self small RNAs. For
example, anti-viral siRNAs bound to AGO2 (the siRNA-
bindin­g AGO protein in flies) bind and destroy viral
RNA without risk of tailing and trimming, because they
have a 2ʹ-O‑methyl-modified 3ʹ end. During viral infec-
tion, non-self-directed siRNAs could be so abundant
© 2013 Macmillan Publishers Limited. All rights reserved
that they inappropriately accumulate in AGO1 com-
plexes. As a consequence, they would not acquire a
protective 2ʹ-O‑methyl modification. Their binding to
fully complementary viral RNAs will instead mark them
as foreign sequences that have inappropriately entered
the miRNA (self) pathway. The tailing and trimmin­g
pathwa­y would then initiate their elimination.
Target RNAs can stabilize miRNAs. In C. elegans, the
5ʹ-to‑3ʹ exoribonuclease XRN‑2 limits miRNA abun-
dance. However, miRNAs in RISC are protected from
XRN‑2 when bound to a partially complementary target
RNA94. Similarly, persistence of siRNAs in cell culture
is enhanced by the presence of a fully complementary
targe­t mRNA95. So, perhaps target binding recruits
miRNA protective factors or translocates miRNAs to
a subcellular location that lacks small RNA-degrading
nucleases such as XRN‑2. Alternatively, target RNAs
may protect miRNAs from a release factor that would
expel them from RISC and expose them to subsequent
destruction94. Supporting this idea, a partially comple-
mentary target RNA also protects worm miRNAs from
destruction by XRN‑1, another 5ʹ-to‑3ʹ exonuclease96.
XRN1 may also degrade miRNAs in animals97.
Diverse strategies to generate isomirs
Although miRBase catalogues miRNAs as single
sequences, the availability of small RNA sequence data
from various organisms, tissues and cell types shows
that miRNAs comprise multiple isoforms (also known
as isomirs ) 4,98–104. Such sequence heterogeneity may
arise from imprecise precursor cropping or dicing,
terminal trimming or the addition of non-templated
nucleotide­s98,103,104 (FIG. 3).
Trimming produces 5ʹ miRNA isoforms. Because the
5ʹ end of a miRNA defines its seed sequence, a single
nucleotide shift at this site will radically alter its target
repertoire. The 5ʹ ends of miRNAs are further con-
strained because AGO loading typically selects for
miRNAs with distinct 5ʹ nucleotides99,102,105–107. Changes
in the 5ʹ end of a miRNA can therefore alter the effi-
ciency of AGO loading. miRNA 5ʹ isoforms comprise
less than 10% of all miRNA reads in humans, flies and
worms4,99,108. Nonetheless, some miRNAs produce two
or more 5ʹ isomirs that repress distinct target mRNAs4,55.
One of the most striking examples is D. melanogaster
miR‑210, which exists as two nearly equally abundant
5ʹ isoforms98.
miRNA 5ʹ isoforms can also arise from differential
processing of paralogous pre-miRNAs, each of which pro-
duces a single miRNA sequence, such as D. melanogaster
pre-miR‑2‑1 and pre-miR‑2‑2 (REF. 99) (FIG.3a).
Other paralogous hairpins, such as human pre-
miR‑133a‑1 and pre-miR‑133a‑2, generate two pre-
dominant isoforms 4. Some extreme examples of
5ʹ  heterogeneity were discovered in mammals and
chicken­s for a set of mirtron­s that contain a long 5ʹ exten-
sion connecting the hairpin to the 5ʹ splice site4,109,110.
After splicing, the 5ʹ extension is trimmed ‘sloppily’ to
produce several mature miRNAs.
 REVIEWS

a 5′ miRNA isoforms
miR-2-1
Paralogous Seed
miRNAs
miR-2-2 5′ 3′

Lariat 5′-to-3′ exonucleolytic

debranching maturation
? +1 –1

Branched mirtron
5′ tailed mirtron

Seed
b Internal miRNA isoforms A-to-I editing
ADAR 5′ 3′
A A
I
c 3′ miRNA isoforms I
Lariat 5′ to 3′ exonucleolytic
debranching maturation

Branched mirtron
3′ tailed mirtron
Seed
Exosome

5′ to 3′ exonucleolytic 5′ 3′
miRNA* maturation
AGO1 Trimming

24 nt 22 nt A
Nibbler Tailing
UU
-2 +2
ZCCH6, ZCCHC11 Dicer
or GLD-2
U U
Pre-miRNA Loqs or Monouridylated
Distributive TRBP miRNA duplex
ZCCHC11 LIN28 DIS3L2
UUU UUU

Processive Pre-miRNA decay

Figure 3 | Mechanisms and consequences of microRNA isoforms.  Precursor microRNAs (pre-miRNAs) and mature
Nature Reviews | Molecular Cell Biology
miRNAs can be modified by the addition, editing or subtraction of nucleotides, resulting in distinct miRNA isoforms.
Such isomirs can be classified as 5ʹ, internal and 3ʹ isoforms. a | 5ʹ miRNA isoforms can be produced by differential
processing of paralogous miRNAs, as exemplified by the miR‑2 family in Drosophila melanogaster or the exonucleolytic
processing of 5ʹ tailed mirtrons. This results in miRNAs with different seed sequences, which are the primary sequence
determinants for target RNA binding. Consequently, different 5ʹ miRNA isoforms regulate different sets of target RNAs.
The identity of the exonuclease that targets 5ʹ tailed mirtrons is not known (indicated by the question mark). b | Internal
miRNA isoforms are rare and almost exclusively generated by editing of adenosine to inosine by the enzyme ADAR
(adenosine deaminase acting on RNA). c | The 3ʹ end of a miRNA shows the greatest heterogeneity, which is a result of
both exonucleolytic trimming and the addition of nucleotides (typically adenine or uridine) to the 3ʹ end (tailing).
Exonucleolytic trimming is required for the efficient processing of 3ʹ tailed mirtrons, as well as ~25% of all miRNAs in flies.
Dicer-1 initially makes these miRNAs as ~24 nucleotide miRNA duplexes. Upon loading into Argonaute 1 (AGO1), such
‘long’ miRNAs are trimmed by the 3ʹ-to‑5ʹ exoribonuclease Nibbler to produce mature ~22 nt miRNAs. In mammals,
monouridylation of selected pre-miRNAs by ZCCHC11, GLD-2 and/or ZCCHC6 enhances substrate recognition and
processing by Dicer. In the case of miRNAs derived from the 3ʹ arm of such pre-miRNAs, monouridylation is propagated
into the mature miRNAs, contributing to its 3ʹ heterogeneity. In the presence of the pre-miRNA-binding protein LIN28,
ZCCHC11 is a processive enzyme, oligouridylating pre-miRNAs that contain a LIN28‑binding motif. Unlike
monouridylation, oligouridylation inhibits Dicer-mediated processing and enhances pre-miRNA decay by the
3ʹ-to‑5ʹ exoribonuclease DIS3L2. Loqs, Loquacious; TRBP, transactivation-response RNA-binding protein.

Mirtrons
Intron-derived precursor
microRNAs excised from
primary miRNAs by the
splicing machinery and a
lariat-debranching enzyme
instead of Drosha.
Internal miRNA isoforms. Modifications that change
the internal sequence of miRNAs are rare and occur at
frequencies that are similar to sequencing errors4,98,111.
However, for a few individual miRNA species, as much
as 80% of all sequence reads for the miRNA contain at
least one base change from the corresponding genomic
sequence4,98,112–114. Such editing events nearly always
correspond to deamination of adenosine to inosine
by ADAR (adenosine deaminase acting on RNA) 115
(FIG. 3b). ADAR enzymes also edit long dsRNAs, pro-
moting their unwinding and preventing them from
triggering RNAi116–118, as well as local double-stranded
structures. Indeed, these local structures may have
evolved to facilitate ADAR-mediated repair of mRNAs

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REVIEWS
Terminal nucleotidyl
transferases
(TNTases). Template-
independent polymerases that
add nucleotides to the 3ʹ ends
of nucleic acids.
480 | AUGUST 2013 | VOLUME 14 www.nature.com/reviews/molcellbio
that lack the appropriate protein-coding sequence.
Because ADAR enzymes are preferentially expressed
in neural tissues of flies and mammals, miRNAs in
the brain are often edited. The biological significance
of miRNA editing outside the seed sequence, however,
remains to be established.
Trimming can produce 3ʹ miRNA isoforms. miRNA iso-
forms often differ at their 3ʹ ends99,100,102,103,108. miRNAs
with a common 5ʹ end but different 3ʹ ends are presumed
to target the same mRNAs, but they may differ in the
extent of target repression or they might have different
half-lifes. Recent studies implicate 3ʹ-to‑5ʹ trimming as
one major source of miRNA 3ʹ heterogeneity.
In D. melanogaster, ~40% of AGO1‑bound miRNAs
are trimmed at their 3ʹ termini, irrespective of whether
they are produced via the canonical or the mirtron path-
way 104. In extreme cases, such as miR‑34 or miR‑317,
four to six variant isoforms accumulate that differ at
their 3ʹ ends104,119,120. This 3ʹ trimming requires the
3ʹ-to‑5ʹ exoribonuclease Nibbler 119,120 (FIG. 3c).
Nibbler mediates trimming of more than a quarter of
all fly miRNAs. It acts predominantly on miRNAs that
arise from pre-miRNAs containing bulges or internal
loops that force Dicer or Drosha to generate an atypically
long product. After the miRNA is loaded into AGO1,
Nibbler trims these long miRNAs into shorter isoforms,
perhaps to ensure that their 3ʹ ends nestle in the AGO1
PAZ domain. Human miRNAs also seem to be trimmed
by a Nibbler-like enzyme120. The C. elegans homologue
of Nibbler, MUT‑7, is required for transposon silencing,
RNAi and co-suppression121–124. But a role for MUT‑7 in
the miRNA pathway has not been reported.
Tailing: a special form of 3ʹ heterogeneity. The addi-
tion of non-templated nucleotides by terminal nucleotidy­l
transferase­s (TNTases) (tailing) gives rise to a special form
of 3ʹ heterogeneity in miRNAs and other types of RNA.
All TNTases are members of the DNA polymerase‑β
superfamily that includes poly(A) polymerase, which
adds the poly(A) tail to mRNAs. TNTases typically add
adenosine or uridine in vivo125. The consequences of tail-
ing can vary. In bacteria, the poly(A) tail triggers mRNA
decay, whereas in eukaryotes it enhances mRNA stability
and translation126. Moreover, uridylation has been impli-
cated in the maturation and turnover of coding and
non‑codin­g RNAs127.
Several studies have recently implicated TNTases in
the regulation of miRNAs, through uridylation of their
3ʹ ends (FIG. 3c). These include ZCCHC6 (also known as
TUT7) and ZCCHC11 (also known as TUT4), which
are both zinc-finger- and CCHC domain-containing
proteins, and GLD2 (also known as TUT2 and PAPD4),
which is a poly(A) polymerase domain-containing-
protein. For example, the TNTase ZCCHC11 oligo­
uridylates pre-let‑7 in mouse embryonic stem cells128,129.
Oligouridylation requires the formation of a ternary
complex, consisting of ZCCHC11, pre-let‑7 and the
pluripotency factor LIN28, which directly binds to a
sequence motif in the loop of pre-let‑7 (REFS 130,131).
Tailing masks the two nucleotide 3ʹ overhang of the
© 2013 Macmillan Publishers Limited. All rights reserved
pre-miRNA and prevents efficient substrate recogni-
tion by Dicer, whereas the oligo(U) tail serves as a decay
signal for the Perlman syndrome exonuclease DIS3L2
(REF. 132).
Surprisingly, in the absence of LIN28, ZCCHC11
and its close relative ZCCHC6 monouridylate a selected
class of pre-miRNAs, including several members of the
pre-let‑7 family (class II pre-miRNAs). Monouridylation
enhances dicing, because it restores the two nucleotide
3ʹ  overhang of pre-miRNAs that were imprecisely
cropped by Drosha133.
Mature miRNAs can also serve as substrates for
TNTases, but the triggers and biological consequence of
this type of modification are less well-established. For
example, the pre-miRNA tailing enzyme ZCCHC11
apparently also adds uridine to mature mammalian
miRNAs such as miR‑26, and this somehow facilitates
cytokine expression134. GLD2 adds one adenosine to
miR‑122, which is the most abundant miRNA in the
liver 135. miR‑122 derives from the 5ʹ arm of pre-miR‑122,
so GLD2 must act on the mature miRNA after dicing.
In mice lacking GLD2, both miR‑122 adenylation and
miR‑122 abundance decrease, suggesting that adenyla-
tion enhances miR‑122 stability. GLD2 adds tails to
several other miRNAs in mammals, although this does
not always increase their stability 136. A protective func-
tion for miRNA adenylation has also been reported in
plants137.
In the absence of the methyltransferase HEN1,
uridylation of mature plant miRNAs by the TNTase
HEN1 SUPPRESSOR 1 (HESO1) triggers miRNA
decay 88,89,138,139. Loss of HESO1 partially restores small
RNA function and suppresses developmental pheno-
types in HEN1 mutant plants138,139 (FIG. 2). Because HEN1
methylates miRNAs before they bind AGO proteins, the
substrates for tailing are inferred to be duplexes.
In the green alga C. reinhardtii, loss of the TNTase
MUT68 reduces small RNA uridylation and increases
miRNA and siRNA abundance140. In vitro, MUT68 tails
synthetic small RNAs, and this promotes their degra-
dation by the catalytic exosome subunit RRP6 (ribo­
somal RNA-processing protein 6). Like loss of MUT68,
loss of RRP6 stabilizes small RNAs in vivo. MUT68 is
also implicated in the degradation of the 5ʹ target RNA
fragments produced by siRNA- or miRNA-directed
RISC cleavage141, which have previously been reported
to serve as substrates for tailing in Arabidopsis thaliana
and mammals142. Perhaps enzymes that can tail either
miRNAs or their targets are both recruited when AGO
proteins bind an RNA.
AGO creates the seed
AGO proteins create a seed in any small RNA sequence
they bind. Mutations in the seed block target cleavage
by plant miRNAs in extracts143 and in vivo144. In addi-
tion, such mutations relieve the repression of highly
complementary mRNAs by miRNAs and siRNAs in
animals145,146, almost certainly because a seed match is
required for high-affinity binding between a miRNA
and its target 147. Structural analyses of archael, bacterial,
yeast and human AGO proteins reveal how AGO creates

the seed79,148–154. Upon AGO binding, the seed bases
are exposed to the solvent and pre-ordered in a quasi-
helical structure that ‘pre-pays’ the entropic penalty
that comes with nucleic acid pairing 151. Structural and
kinetic data suggest that the seed nucleates binding to
complementary sequences in the target RNA (the seed
match) and that pairing of the target with sequences 3ʹ to
the seed can only occur subsequently, after a structural
rearrangemen­t of the AGO protein79,147,150,152,155.
This nucleation theory was recently modified after
the discovery that a set of miR‑124 targets in the mouse
brain seem to lack full seed complementarity. Instead,
they contain one ‘bulged’ nucleotide across from posi-
tions 5 and 6 of the miRNA seed156 (FIG. 4). In these cases,
an initial seed–target interaction across five nucleotides
has been proposed to rearrange into a six nucleotid­e
helix containing one additional unpaired (that is, flipped
out or bulged) target base. Such sites constitute more
than 15% of all AGO-bound sequences in the mouse
brain, are evolutionary conserved and mediate target
mRNA repression156. These studies are consistent with
in vitro analyses of AGO in the eubacterium Thermus
thermophilus, which tolerates a flipped out base in
the seed-matching sequences of its target 150, as well
as thermo­dynamic studies on the recognition of let‑7
target­s in worms157.
Pairing between a target and miRNA bases that are 3ʹ
to the seed — that is, the ‘supplementary’ base pairs from
miRNA position 13 to 16 — can decrease the off rate
(koff) of RISC from a target RNA, which may explain why
such additional pairing can enhance target repression64,147
(FIG. 4e). Moreover, features other than base pairing can
have a substantial effect on target regulation and will
contribute to the quality of a miRNA-binding site. These
include: an adenosine across from the first nucleotide of
the miRNA70,158; the accessibility of a target site64,159–161;
and the position of a target site within a 3ʹ UTR64. mRNA-
binding proteins add an additional layer of complexity,
as they can compete with and perhap­s even enhance
miRNA binding 162–169.
Alternative modes of miRNA target recognition have
been reported, but these seem to have a modest impact
on gene regulation: fewer than 10% of all target sites are
predicted to include base pairs that compensate for an
incomplete seed match, and even fewer sites lack seed
matches altogether 63,170 (FIG. 4e). miRNAs can also con-
tain sequence motifs that regulate their stability or intra-
cellular localization (BOX 1). Nonetheless, the association
of a seed sequence with the target mRNA remains the
predominant mechanism for target recognition.
The great miRNA function debate
Despite a plethora of high-quality studies examining the
biochemistry, biology and genomics of miRNA-directed
mRNA regulation, how miRNAs repress or activate gene
expression in animals remains controversial. There is
general agreement that miRNAs regulate gene expres-
sion post-transcriptionally, but several mechanisms have
been proposed to explain how, ranging from repression
of translational initiation or elongation to acceleration of
general mRNA decay processes (FIG. 4c,d).
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 14 | AUGUST 2013 | 481
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
How do miRNAs regulate mRNA expression? Some
AGO proteins cleave highly complementary target
RNAs, a process that is catalysed by their RNase H‑like
PIWI domain, which positions a pair of Mg 2+ ions at
the scissile phosphate. This endonucleolytic mechanism
derives from the ancestral RNAi pathway, in which target
‘slicing’ provides an anti-viral and anti-transposon
defence.
In plants, highly complementary miRNA target
sites trigger mRNA cleavage143,171–176, but at least some
miRNAs can block translation177,178 (FIG. 4). By contrast,
just a few miRNA–target pairs in mammals have suf-
ficient complementarity to direct AGO2 to cleave the
target 170,179–181. Even for these atypical examples, the con-
tribution of miRNA-directed target cleavage to the over-
all decrease in target mRNA abundance is unknown.
Moreover, three of the four human AGO clade proteins
are catalytically inactive; only a miRNA bound to AGO2
can cleave a highly complementary target RNA. This
suggests that AGO proteins more often mediate target
gene expression control through a mechanism that is
independent of RNA endonucleolytic cleavage.
The overwhelming majority of miRNAs recognize
their targets solely through their seed sequence, and such
partial complementarity does not permit target cleavage
even when the miRNA is bound to AGO2. Nonetheless,
animal miRNAs typically trigger destruction of the
mRNAs they bind182–190. Quantitative high-throughput
sequencing and proteomics methods for measuring
mRNA and protein abundance as well as new methods
that measure ribosome occupancy of mRNAs have only
fuelled the debate on how miRNAs regulate their targets.
Ribosome profiling experiments in mice suggest
that mRNA destruction by a process distinct from
endonucleolytic cleavage, rather than translational
repression, explains the overwhelming majority
(>84%) of miRNA-directed repression in mammals188.
Ribosome profiling quantitatively determines the posi-
tions of ribosomes on cellular mRNAs with codon-
level resolution for the entire transcriptome191. Such
data have reinforced the view that most of the decrease
in steady-state protein abundance that is observed
during miRNA-mediated repression reflects mRNA
decay 188,189,192. Nonetheless, a small but significant frac-
tion (11–16%) of miRNA repression seems to derive
from a block in translation190.
Although mRNA destruction is necessarily mutually
exclusive with translational repression, studies in zebrafish
suggest that translational repression can precede mRNA
degradation. Zebrafish miR‑430 is the predominant
miRNA expressed at the onset of zygotic transcription.
miR‑430 clears maternal mRNAs from the embryo, a
process that is prevented in mutants lacking both mater-
nal and zygotic Dicer 185,193. Ribosome profiling of wild-
type and Dicer mutant fish shows that miR‑430 initially
reduces ribosome occupancy of target mRNAs — which
represses their translation — and then, two hours later,
triggers mRNA destruction193. The initial decrease pri-
marily reflects a change in the rate of translational initia-
tion, a mechanism of miRNA action previously observed
for fly AGO1 in  vitro 194–196 and in cell extracts197,198.
REVIEWS

a Endonucleolytic cleavage b
AGO m7Gppp Plants (frequent) 10,11
(n)AAAA miR-171 5′-UGAUUGAGCCGCGCCAAUAUC-3′
SCL6 mRNA 3′-...ACUAACUCGGCGCGGUUAUAG...-5′

(n) AAAA Animals (rare) 10,11

miR-196a 5′-UAGGUAGUUUCAUGUUGUUGGG-3′
UUUU
HoxB8 3′ UTR 3′-...AUCCGUCAAAGUACAACAACCU...-5′
XRN1 or XRN4
Exosome

c Translational repression
Block to translation e
initiation Plants (rare?)
miR-172a/c 5′-AGAAUCUUGAUGAUGCUGCAU-3′
SCL6 mRNA 3′-...UCUUAGGACUACUACGACGUC...-5′
m7Gppp
(n)AAAA
ORF
Animals (canonical seed match site; most frequent)
2 8
lin-4 5′-UCCCUGAGACCUCAAGUGUGA-3′

Change in repressive mechanism over time?

lin-14 3′ UTR 3′-...AGGGACUCAACCUAAUUUUCU...-5′
AA

A Deadenylation
A Animals (G-bulge site; less frequent?)
Recruitment of 2 8
translation blockers miR-124 5′-UAAGG-CACGCGGUGAAUGCC-3′
Mink1 3′ UTR 3′-...GUUCCGGUGAUGUAACUCUUU...-5′

(n)AAAA
Animals (3′ supplementary site; less frequent?)
2 7 13 16
miR-2 5′-UAUCACAGCCAGCUUUGAGGAGC-3′
grim 3′ UTR 3′-...UUAGUGUUACGCGAAACUAACUC...-5′
d mRNA turnover Decapping and
5′-to-3′ decay
Animals (3′ compensatory site; rare)
2 8
let-7 5′-UGAG-GUAGUA-GGUUGUAUAGUU-3′
AA
lin-41 3′ UTR 3′-...ACUCACAUCUUGCCAACAUAUUUU...-5′
A Deadenylation
A

Figure 4 | microRNA function in plants and animals.  a | Most plant microRNAs (miRNAs) and a few animal miRNAs
direct endonucleolytic cleavage (slicing) of their mRNA targets. The 5ʹ‑to‑3ʹ exoribonuclease XRN4
Nature Reviews in plants and
| Molecular CellXRN1
Biology
in animals, together with the major cellular 3ʹ‑to‑5ʹ exonucleolytic complex, the exosome, subsequently degrade the
sliced mRNA fragments. The 3ʹ end of the 5ʹ cleavage product is frequently uridylated, perhaps to mark these fragments
for decay. b | miRNA-directed endonucleolytic cleavage of mRNAs requires extensive complementarity between the
miRNA and its target site (representative examples in plants and mammals are shown). Endonucleolytic cleavage occurs
at the phosphodiester bonds across from nucleotides 10 and 11 of the miRNA, counted from the miRNA 5ʹ end. Although
slicing seems to be the dominant mode of action for miRNAs in plants, highly complementary sites in the transcriptome
of animals are rare. c | In animals, miRNAs were originally proposed to repress translation of an open reading frame (ORF).
Biochemical studies have suggested that miRNAs have a role in blocking translational initiation, in poly(A) tail
shortening  or in the recruitment of protein cofactors that can interfere with translation. d | In many cells and tissues,
miRNA-directed translational repression is indistinguishable from mRNA destruction via decapping and 5ʹ‑to‑3ʹ decay.
This has led to the suggestion that miRNAs directly target mRNAs for decay. Another possibility is that the inhibition of
translation by miRNAs (part c) triggers subsequent mRNA decay, and the temporal delay between these two effects can
vary depending on the surveillance mechanisms in place in particular cellular contexts (depicted by blue arrow on the
right). e | Like animal miRNAs, plant miRNAs may regulate target mRNAs via mechanisms other than endonucleolytic
cleavage. But the underlying miRNA–target RNA interaction (for example, miR‑172–SCL6) is indistinguishable from sites
that trigger cleavage (part b). In contrast to plants, animal miRNAs are almost always imperfectly complementary to the
target mRNAs they regulate. The seed sequence (miRNA nucleotides 2 to 8; sometimes interrupted by a specific
G‑bulge) is the major determinant for target binding and often suffices to trigger mRNA repression. Additional pairing
of miRNA nucleotides 12 to 16 can sometimes bolster seed binding (3ʹ supplementary sites). Only in rare cases can an
imperfectly matching seed sequence be compensated for by extensive complementarity between the miRNA 3ʹ region
and the target site (3ʹ compensatory sites). Binding of animal miRNAs to partially complementary target sites generally
results in translational inhibition and/or mRNA decay via the mechanisms shown in part c and part d. HoxB8, homeobox B8;
UTR, untranslated region.

482 | AUGUST 2013 | VOLUME 14 www.nature.com/reviews/molcellbio

© 2013 Macmillan Publishers Limited. All rights reserved


Box 1 | microRNA sequence motifs
Sequence motifs within a microRNA (miRNA) can direct its localization to particular
sites in a cell or enhance its turnover. For example, a hexamer motif in miR‑29b triggers
its nuclear import241. Nuclear miR‑29b may regulate gene expression in the nucleus or,
alternatively, the nuclear import of miR‑29b may alter target gene expression in the
cytosol by sequestering the miRNA and effectively decreasing its concentration in
the cytoplasm. This hexamer motif is necessary and sufficient for nuclear import,
as insertion of this sequence into another miRNA directs its nuclear import.
Several sequence motifs have been linked to miRNA turnover. In the absence of such
motifs, miRNAs are long-lived, with half-lives as long as 5 days242 or even 2 weeks243.
For example, a seven nucleotide motif at the 3ʹ end of miR‑382 triggers its decay in
HEK293 cells97. Furthermore, destruction of miR‑16 family miRNAs upon re‑entry into
the cell cycle after serum starvation relies on both the seed sequence and a sequence
at the 3ʹ end of the miRNAs244. Uridylation of transiently transfected miR‑29b and
miR‑29c at positions 9–11 elicits their rapid turnover in HeLa cells, a phenomenon that
seems to be conserved in the fly miRNA bantam245. For miR‑29b and miR‑29c, the
miRNA–miRNA* duplex seems to be targeted for degradation rather than the mature
Argonaute (AGO)-bound miRNA. The proteins that recognize the sequence motifs
controlling miRNA localization or stability and whether such recognition occurs while
the miRNA is still bound to AGO are not yet known.
The timing of mRNA poly(A) tail shortening, which was
previously proposed to explain miR‑430‑directed transla-
tional repression in the same model system185, is similar to
the effects on mRNA decay but not to the initial decrease
in translational initiation193. Together with previous bio-
chemical studies198–201 and more recent kinetic analysis
in a D. melanogaster cell line202, these studies point to a
testable, unifying hypothesis: miRNAs initially repress
mRNA translation and later trigger mRNA decay, per-
haps in response to the block to translation rather than
any specific property of miRNAs or AGO proteins. The
timing of decay might vary depending on the context:
in cells with robust surveillance mechanisms, mRNAs
bound by miRNAs would be rapidly degraded, whereas in
early embryos, translationally repressed mRNAs would be
stable. Mutations in the mRNA surveillance or degrada-
tion machinery should prove useful in testing these ideas.
We note that all forms of this ‘translational-repression-
triggers-mRNA-decay’ hypothesis imply biochemical
coupling between the rate of translation and the stability
of an mRNA. Proof of such coupling will require rigorous,
quantitative in vivo measurements of the rates of transla-
tion and decay for mRNAs targeted by miRNAs in the
presence and absence of miRNA function.
miRNAs turn me on? miRNAs generally repress gene
expression, but they have also been linked to gene acti-
vation. Often, the mechanism of activation is indirect,
with repression of a repressor leading to increased
expression of specific transcripts. One such case occurs
during neuronal differentiation in frogs, chickens and
mammals203, in which miR‑128 in the brain targets
components of the nonsense-mediated decay machin-
ery and thereby increases the abundance of mRNAs
involved in neuronal differentiation and brain function.
A second example may be the activation of trans­
cription by miR‑373 (REF.  204) . In other contexts,
repression of non-coding transcripts that cross the
promoters of overlapping genes have been reported to
increase the abundance of the sense gene transcript205,206.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 14 | AUGUST 2013 | 483
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
Although the precise mechanism by which overlap-
ping non-coding transcripts decrease gene expression
remains unknown, miR‑373 may similarly destabilize or
block the function of non-coding transcripts that cross
the promoter regions of protein-coding genes.
The requirement for miR‑122 in hepatitis C virus
replication is a particularly intriguing example of activa-
tion by a miRNA: binding of miR‑122 to complementary
sites in the 5ʹ end of the virus genome is required for effi-
cient viral replication and enhances internal ribosome
entry (IRES) site-directed translation207. The mechanism
of this activation is not known, nor is it clear whether
miRNAs have similar effects on viral gene expression
as they do on mammalian mRNAs. Nonetheless, the
finding that a readily inhibited host factor is required
for viral replication makes miR‑122 one of the most
promisin­g drug targets for treating hepatitis C208,209.
A more unusual and direct form of miRNA activa-
tion of gene expression has been described following cell
cycle arrest. Upon serum starvation, miR‑369‑3 binds
an AU‑rich element in the 3ʹ UTR of the gene encodin­g
tumour necrosis factor (TNF). miR‑369‑3 normally
directs repression but, in these specific conditions,
it seems to promote translation210,211. A similar phenom-
enon has also been observed for the translational activa-
tion of ribosomal protein genes, although miR‑10a was
proposed to bind non-seed-matching sites in the 5ʹ UTR
of mRNAs212.
Apostasy or prophecy? Not only are the mechanisms
by which miRNAs repress gene expression debated but
also who represses whom. The crux of the issue is the
seemingly modest effect that miRNAs have on the vast
majority of mRNAs they target. A generally accepted
hypothesis posits that many miRNAs ‘tune’ the expres-
sion of most targets, with only a small number of target­s
experiencing a large change in mRNA or protein abun-
dance63. The biological importance of such tuning is
thought to emerge in specific environmental conditions
that are not typically encountered by animals reared in a
laboratory. Supporting this view, the role of miR‑7 in
ensuring correct patterning of the D. melanogaster eye
is only apparent in sensitized genetic backgrounds or
when the fly experiences ‘wild swings’ in temperature
during eye development 213. Similarly, the loss of indi-
vidual miRNA­s or miRNA families in C. elegans rarely
produces an obvious phenotypic defect214.
An alternative view proposes that most ‘tuning targets’
are actually ‘titrating targets’ that moderate the effective
concentration of the miRNA available to bind a smaller
set of highly regulated targets215. The concept of titrat-
ing sites is supported by the finding that artificial, long,
highly abundant RNAs containing miRNA-binding sites
can act as competitive inhibitors of specific miRNAs.
Indeed, the first specific miRNA inhibitors were synthetic
anti-miRNA oligonucleotides216,217. Similarly, transgenic
or transfected genes containing multiple miRNA-binding
sites can inhibit the function of miRNAs in animals218–220.
Such miRNA ‘sponges’ typically contain non-natural
miRNA-binding sites that are designed with central
mismatche­s to block target cleavage.
REVIEWS

Biologically irrelevant Biologically relevant

[miRNA]<<Kd [miRNA]>>Kd
AGO AGO

m7Gppp m7Gppp
(n)AAAA
ORF (n)AAAA
ORF
No occupancy [Target sites] < [miRNA] [Target sites] > [miRNA]
Frequent? Rare?

Strong regulation Weak regulation

Figure 5 | A quantitative framework for microRNA function.  In this model, the relative abundance of microRNAs
(miRNAs) and their targets inside the cell dictates the regulatory outcome. At concentrations
Nature Reviewsbelow the affinity
| Molecular of Biology
Cell
Argonaute (AGO)-bound miRNAs for their targets (Kd), occupancy of miRNAs at mRNA targets is negligible and, thus, the
biological function of these miRNAs is irrelevant. If present at concentrations above the Kd (this is typically true at least
for the most abundant miRNAs in a given cell type or tissue), virtually every target site will be bound by a miRNA. In this
case, the cumulative abundance of all target sites in the cell, relative to the abundance of the miRNA, will determine the
extent to which each individual target mRNA is regulated. Fewer total target sites than the corresponding miRNA will
prompt a larger extent of regulation compared with miRNAs that are less abundant than their target sites.

miRNA sponges occur naturally in plants and act to
competitively inhibit miRNA repression of other targets
in a phenomenon termed target mimicry 221. For exam-
ple, phosphate starvation in A. thaliana induces the
expression of both miR‑399 and the non-coding RNA
INDUCED BY PHOSPHATE STARVATION 1 (IPS1).
IPS1 binds miR‑399 through a site that contains cen-
tral mismatches that protect IPS1 from AGO-catalysed
cleavage. IPS1 thereby reduces the amount of miR‑399
available to repress its target mRNA PHOSPHATE 2.
Transgenic non-cleavable miRNA sponges modelled after
IPS1 inhibit individual miRNAs in plants221,222.
Notably, the titrating targets model leaves unexplained
the apparent conservation of miRNA-binding sites in
titrating targets: how would so many sites, each of which
contributes just a little to reducing the free pool of the
miRNA, be under positive evolutionary selection? Should
such sites not come and go over evolutionary time, with
only their number and not their locations within any
specific mRNA being conserved? A more speculative
and thermodynamically implausible version of this idea
proposes that individual target RNAs — not the collec-
tive abundance of miRNA seed matches — can titrate
the amount of miRNA that is available to repress their
targets223. For example, protein-coding and non-coding
transcripts have been predicted to regulate the dosage-
sensitive tumour suppressor gene PTEN by competing
with the PTEN transcript for binding to a similar set of
miRNAs224. A decrease or loss of competing transcripts,
for example derived from the pseudogene PTENP1 or
the protein-coding gene zinc-finger E‑box-binding
homeobox 2 (ZEB2), would increase the amount of
miRNAs available to repress PTEN and hence accelerate
tumorigenesis225–228.
Among the objections to this idea are the findings
that many miRNAs are more abundant than the sum
of their individual mRNA targets and that the concen-
tration of miRNAs in vivo are probably far greater than
their dissociation constant (Kd) values for seed match
sites 147. Nematode and human miRNAs can reach
50,000 molecules per cell, with some being as abundant
as the spliceosomal U6 small nuclear RNA (snRNA)229,230.
By contrast, the 5,000 most abundant mRNA species in
nematodes are thought to be present at just ~100 mol-
ecules each per cell229. Many miRNAs therefore out-
number any single mRNA target by as much as 500‑fold.
However, each animal miRNA has the potential to regu-
late hundreds of mRNAs, some of which contain more
than one miRNA‑binding site. Consequently, the num-
ber of some miRNAs may be similar to the number of
miRNA‑bindin­g sites, suggesting that a model in which
a miRNA is regulated by numerous titrating targets is
biochemically more feasible than one in which individual
target mRNAs are key. Moreover, computer simulations
using experimentally measured Kd and koff values suggest
that only miRNAs that are least likely to provide meaning-
ful target repression in a given cell type could be subject to
competition by highly abundant, individual non-coding
RNAs or mRNAs bearing miRNA-binding sites147.
A quantitative framework for miRNA and target RNA
function. The biochemical and biophysical properties of
AGO-bound miRNAs provide a quantitative framework
for understanding the reciprocal function of miRNAs and
their targets according to their relative abundance inside
the cell (FIG. 5). When a miRNA is dilute, that is, at concen-
trations lower than the affinity of AGO-bound miRNA­s
for their targets, the overall impact of the miRNA on
gene expression is expected to be negligible147. This pre-
diction is supported by miRNA sensor assays in cultured
monocytes, which revealed that only the most abundant
miRNAs are able to change the expression of a reporter
construct containing a miRNA-binding site231.
Highly abundant miRNAs (present at intracellular
concentrations that vastly exceed the Kd for miRNA
binding to a typical binding site on an mRNA target) are

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© 2013 Macmillan Publishers Limited. All rights reserved

 REVIEWS

predicted to be bound to target mRNA. The extent of reg-
ulation, that is, the number of sites on each target mRNA
occupied by a miRNA, is then determined by the overall
abundance of target sites in the transcriptome. In this
case, significant regulation of miRNA activity through
competitive transcripts is in principle possible, but only
if the overall abundance of the competitor is much higher
than the total number of miRNAs. Experimentally, this
can be achieved by either expressing artificially high lev-
els of sponge transcripts220,232–234 or by vastly increasing
the number of miRNA-binding sites in each transcript219.
For endogenous mRNAs or non-coding transcripts,
it is unlikely that sufficiently high concentrations can
be achieved to allow competitive inhibition of abundant
miRNAs, particularly because miRNA binding to the
competitor transcripts should decrease their steady-state
abundance via one of the mechanisms highlighted above.
Nevertheless, miRNA decay induced by viral infection
suggests that mechanisms exist that efficiently inhibit
miRNA function by triggering their decay upon inter­
action with target RNAs235–237, perhaps because enzymatic
degradation and not simple competition renders this pro-
cess efficient72–74,236. Because viruses only contribute the
target RNA signal for miRNA decay, and the molecular
machinery seems to be provided by the host cell itself, it
is tempting to speculate that this process may be used to
regulate miRNA function through cellular transcripts in
uninfected cells, but biological examples of this have not
yet been identified.
Squaring the circle. An unexpected, natural solution to
the competitive miRNA inhibitor paradox seems to have
now been identified in the form of previously overlooked
circular RNAs (circRNAs)238–240. circRNAs are generated
by head‑to‑tail splicing of exons. The human circRNA,
CDR1as (antisense to the cerebellar degeneration-related
protein 1), contains 63 conserved sites with imperfect
complementarity to miR‑7 and is proposed to function
as a ‘super-sponge’ to regulate miR‑7 function in the neu-
ronal tissues in which both the miRNA and the circRNA
are co‑expressed. Although the biological function and
the general scope of this type of regulation are unclear,
ectopic expression of CDR1as in zebrafish, which natu-
rally produce miR‑7 but not CDR1as, impairs midbrain
development, recapitulating the loss‑of‑function pheno-
type of miR‑7 (REF. 240). The high abundance and strik-
ingly high numbers of miRNA-binding sites in CDR1as
are consistent with its proposed function in miRNA inhi-
bition. More intriguingly, its circular architecture might
prevent its degradation by miRNAs, particularly if such
miRNA-directed destruction requires ribosome binding
or recruitment of exonucleases.
Conclusions and perspectives
This year marks the twentieth anniversary of the discovery
of miRNAs1,2. In 1993, it would have been hard to imagine
the extent and complexity of miRNA-mediated regulation
of gene expression. Those studying these tiny riboregu-
lators can deservedly be proud of our current catalogue
of miRNAs, their mRNA targets and mechanisms of
biogenesis and sequence diversification. Although many
details surely remain to be discovered, what we most lack
is a deeper understanding of the biological functions of
miRNAs. Are unifying principles and deeper explana-
tions for the role that miRNAs have in coordinating gene
expression during development and environmental expe-
rience to be found? Or does much of what we now view
as miRNA-mediated regulation or seemingly purposeful
mechanisms to diversify miRNA sequences simply cor-
respond to neutral experiments in evolution, the results
of which we will be unable to observe? We believe that the
answers to these and other questions about miRNAs lie in
rigorous computational, biochemical and genetic tests of
quantitative models.

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Acknowledgements
The authors thank the members of the Zamore and Ameres
laboratories for helpful discussions and comments. The
Ameres laboratory is funded by the Austrian Academy of
Sciences and the Austrian Federal Ministry of Economy,
Family and Youth (BMFWJ).
Competing interests statement
The authors declare no competing financial interests.
FURTHER INFORMATION
Phillip D. Zamore’s homepage:
http://www.umassmed.edu/zamore/index.aspx
Stefan L. Ameres’ homepage:
http://www.imba.oeaw.ac.at/research/stefan-ameres
The microRNA database: http://www.mirbase.org
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