Mol Genet Genomics
DOI 10.1007/s00438-010-0556-1

R EV IE W

Many ways to generate microRNA-like small RNAs:
non-canonical pathways for microRNA production
Keita Miyoshi · Tomohiro Miyoshi · Haruhiko Siomi

Received: 2 June 2010 / Accepted: 22 June 2010
© Springer-Verlag 2010

Abstract MicroRNAs (miRNAs) are an abundant classof small non-coding RNAs that collectively regulate theexpression of a large number of mRNAs by either promoting destabilization or repressing translation, or both. Therefore, they play a major role in shaping the transcriptomes
and proteomes of eukaryotic organisms. Typically, animal
miRNAs are produced from long primary transcripts with
one or more of hairpin structures by two sequential processing reactions: one by Drosha in the nucleus and the other byDicer in the cytoplasm. However, deviations from this paradigm have been observed: subclasses of miRNAs, which
only partially meet the classical deWnition of a miRNA, arederived by alternative biogenesis pathways, thereby providing an additional level of complexity to miRNA-dependent
regulation of gene expression.
Keywords miRNA · Drosha · Dicer · Argonaute ·snoRNA · tRNA

Introduction
MicroRNAs (miRNAs) are a family of small regulatory
RNAs that post-transcriptionally control the expression ofCommunicated by J. Graw.
K. Miyoshi · T. Miyoshi · H. Siomi (&)
Department of Molecular Biology,
Keio University School of Medicine,
35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japane-mail: awa403@sc.itc.keio.ac.jp
URL: http://web.sc.itc.keio.ac.jp/dmb/sindex.html
K. Miyoshi
e-mail: mkeita@a3.keio.jp

speciWc genes by base-pairing, typically to the 3⬘-untranslated regions (3⬘-UTRs) of target messenger RNAs(mRNAs) to direct a reduction in their translation and/or
stability (Filipowicz et al. 2008). They are involved in a
broad range of developmental, cell diVerentiation andcell maintenance functions (Bushati and Cohen 2007), andmiRNA perturbations are, therefore, closely linked tohuman diseases, such as cancer (Chang and Mendell 2007;
Garzon et al. 2009). There are hundreds of unique miRNAs
in a given species, and computational and experimental
approaches indicate that individual miRNAs can base-pairand selectively regulate a plethora (probably several hundreds) of diVerent target mRNAs (Bartel 2009; Friedman
et al. 2009). Therefore, miRNAs greatly inXuence the spatio-temporal, global expression patterns in eukaryoticorganisms (Baek et al. 2008; Bushati and Cohen 2007; Limet al. 2005; Selbach et al. 2008). Importantly, recent datacollectively suggest a correlation between miRNA diversity
and morphological complexity as measured, for instance,by the total number of neurons in an organism (Grimson
et al. 2008; Technau 2008).
miRNAs are approximately 22 nucleotides (nt) and originates from a precursor with a characteristic hairpin orstem-loop structure that does not contain large internalloops or bulges (Berezikov et al. 2006). miRNAs are 5⬘phosphorylated and 2⬘-3⬘-hydroxylated and are typicallyprocessed through two sequential steps in animal cells: theWrst in the nucleus by an RNase III enzyme termed Drosha,
and the second in the cytoplasm by another RNase III
enzyme termed Dicer. These enzymes trim the genomeencoded stem-loop precursor into the mature miRNA(Carthew and Sontheimer 2009; Kim et al. 2009; Siomi andSiomi 2009). In principle, this process could be regulated at
multiple levels. Indeed recent studies hint that each one of
the miRNA processing steps is regulated, and that each123

Mol Genet Genomics

miRNA is probably regulated independently, thereby providing an additional level of complexity to the miRNAdependent regulation of gene expression (Siomi and Siomi2010). Mature miRNA associates with a member of the
Argonaute (AGO) family of proteins to form the core of theRNA-induced silencing complex (RISC: also referred to as
the miRISC) and are then poised to interact and repress
their target mRNAs.
Recent analyses of massive amounts of data produced by
next generation sequencing technologies, also known as
deep sequencing or high-throughput sequencing, frommany animal cell types and tissues, have revealed subclasses of miRNA species that are derived by alternative biogenesis pathways and only partially meet the classicaldeWnition. It is important to bear in mind that in many ofthese analyses, sequences that met at least two operational
criteria, the expression of a »22-nt form and the presenceof a hairpin precursor (Berezikov et al. 2006), were classiWed as non-canonical miRNAs. Here, we review recent
progress in our understanding of the non-canonicalmechanisms of miRNA biogenesis. Although much of theevidence discussed here refers to mammalian systems, italso includes a broader perspective from other systems.

Canonical miRNA biogenesis
miRNA genes are transcribed typically by RNA polymerase II (pol II) to generate a long primary miRNA (primiRNA), which is characterized by a hairpin or fold-backstructure with a »32-nt long imperfectly base-paired stem
and a terminal loop. The pri-miRNA is 5⬘-capped, spliced
and polyadenylated, and can form two or more hairpins,
each containing a distinct mature miRNA species (Carthew
and Sontheimer 2009; Kim et al. 2009). Long pri-miRNAs
driven by pol II can be either coding (mRNA precursors) or
non-coding (Kim et al. 2009). Thus, maturation of miRNAs
from pri-miRNAs involves the precise recognition and theaccurate excision and release of functional mature miRNAs. In animal cells, pri-miRNAs undergo two-step processing: Wrst in the nucleus and second in the cytoplasm(Carthew and Sontheimer 2009; Kim et al. 2009; Siomi andSiomi 2009) (Fig. 1a). Within the nucleus, hairpin structures within the pri-miRNA are recognized by a multiprotein complex called the Microprocessor, of which the corecomponents are the RNase III Drosha and the doublestranded RNA-binding domain (dsRBD) protein DGCR8/
Pasha (Denli et al. 2004; Gregory et al. 2004). DGCR8
directs Drosha to cleave the pri-miRNA at the base of thehairpin, resulting in a precursor miRNA (pre-miRNA) of
60–70 nt in length (Han et al. 2006). Exportin-5 recognizes
the 2-nt 3⬘-overhang, characteristic of RNase III-mediatedcleavage, and transports the pre-miRNA into the cytoplasm

123

via a Ran-GTP-dependent mechanism (Kim et al. 2009;
Okada et al. 2009).
Within the cytoplasm, a second RNase III, Dicer, whichpairs with the dsRBD proteins TRBP/PACT/Loquacious(Loqs), cleaves the pre-miRNA to release a »22 nt miRNA
duplex, referred to as the miRNA/miRNA* duplex(Chendrimada et al. 2005; Förstemann et al. 2005; Jiang
et al. 2005; Lee et al. 2006; Saito et al. 2005) (Fig. 1a). These
are 5⬘-phosphorylated, 2⬘-3⬘-hydroxylated small RNAs, aconsequence of the sequential processing by the RNaseIII
enzymes Drosha and Dicer. One strand of the duplex, typically with a relatively lower stability of base-pairing at the5⬘-end (“the thermodynamic asymmetry rule”), is destined
to become the mature guide miRNA (Khvorova et al. 2003;
Schwarz et al. 2003). This strand selectively associates with
an AGO protein to form the core of the miRISC. The phosphorylated 5⬘-end of the miRNA guide strand is critical forthe interaction with the AGO protein (Ma et al. 2005;
Parker et al. 2005; Parker 2010). Once incorporated into themiRISC, the miRNA guides it to complementary sites typically but not always within the 3⬘-UTRs of the target
mRNAs, to mediate repression of that target message(Bartel 2009). Most of the pairing speciWcity for target recognition is provided by the 5⬘-proximal “seed” region(nucleotides 2–8) of the miRNA (Bartel 2009). In somecases, miRNA repression can be reversed (Bhattacharyya
et al. 2006), and the miRISC can even activate expressionof the target (Vasudevan et al. 2007; Cordes et al. 2009).
How the miRISC represses or activates the expression of
speciWc genes appears to be context-dependent and is the
subject of ongoing debate (Brodersen and Voinnet 2009;
Filipowicz et al. 2008; Gu et al. 2009).
The passenger (opposite) strand of the mature miRNA,referred to as miRNA*, is excluded from the miRISC and istypically degraded. However, recent deep sequencing
eVorts indicate that a large number of miRNA*s are associated with AGOs and that the relative expression levels of
the two strands vary widely among tissues (Czech et al.
2009; Hu et al. 2009; Okamura et al. 2008, 2009; Seitz
et al. 2008). In Xies, although miRNAs are typically loaded
onto AGO1, the sorting of miRNAs and miRNA*s into
diVerent AGO complexes (AGO1 and AGO2 complexes)
occurs in some cases. The 5⬘-ends of both miRNAs andmiRNA* strands are typically more precisely deWned thantheir 3⬘-ends, further implying that miRNA* sequences areunder selective pressure to establish a unique seed sequenceat the AGO loading step, implying that many miRNA*
sequences are under evolutionary pressure to maintain theirseed sequences (Seitz et al. 2008). In addition, miRNA*sseem to impact on the evolution of 3⬘ UTRs (Okamura
et al. 2008), suggesting that they have a true regulatory
function. Thus, these Wndings together suggest that, in addition to the thermodynamic asymmetry rule, additional

Mol Genet Genomics

Fig. 1 Canonical and non-canonical pathways of miRNA biogenesis.
a Canonical pathway of miRNA biogenesis in animals. miRNAs aretranscribed from the genome as long primary transcripts. Pri-miRNAs
form hairpin structures, and are processed to about 60–70 nt precursor
microRNAs (pre-miRNAs) by the Microprocessor (Drosha-DGCR8/
Pasha complex) in the nucleus. After being exported by Exportin-5,
pre-miRNAs are further processed to a »22 nt miRNA/miRNA*
duplex by the Dicer1-TRBP/PACT/Loqs complex in the cytoplasm.
Finally, mature miRNAs are loaded onto Argonaute proteins. b Mirtronderived pathway. Short introns with hairpin potential, termed mirtrons,
can be spliced and debranched into pre-miRNA hairpin mimics thatbypass Drosha cleavage. Debranched mirtrons access the canonicalmiRNA pathway during nuclear export, and are then cleaved by Dicer
and loaded onto Argonaute proteins. c snoRNA-derived pathway. The
processing of snoRNAs to miRNAs requires Dicer activity but is independent of Drosha/DGCR8. d shRNA-derived pathway. The predictedsecondary structure of the presumed mir-1980 precursor is shown. The
processing may be similar to that of the snoRNA-derived pathway.

e tRNA-derived pathway. Predicted secondary structures for the tRNAcloverleaf structure and alternative shRNA hold. f tRNase Z-dependent pathway. miRNAs encoded by MHV68 derived from one arm of
one out of two proposed pre-miRNA-like stem loops located 3⬘ of thetRNA-like structure. The processing of the pre-miRNA is dependent
on tRNase Z but is independent of Drosha in the nucleus. After nuclear
export, pre-miRNA is processed by Dicer. g miRNAs derived from aterminal hairpin of an endo-siRNA precursor. Endo-siRNA precursors
are processed by the Dicer2 complex to yield pre-miRNA-like terminal
hairpins, which are then processed by the Dicer1 complex. Then, thesesmall RNAs are loaded onto AGO1 to form the core protein of the
miRISC. h Dicer-independent pathway. Although Drosha cleavage
proceeds normally, the Dicer step is skipped and the pre-miR-451 isloaded directly into Ago2. i Dicer-independent, tRNase Z-dependent
pathway. A 3⬘-trailer fragment of a precursor tRNA is released by acleavage by tRNase Z. Organisms that represent each pathway (b–i)
are indicated at the bottom of the panels. D.m. Drosophila melanogaster, G. lamblia Giardia lamblia

strand selection mechanisms operate on the miRNA duplex
in animal cells, and in such cases both strands are likely to
be functional (Okamura and Lai 2008). Other determinants
for strand selection and partitioning into diVerent AGO
complexes include the structure of the miRNA/miRNA*
duplex (e.g., the positions of mismatches) (Tomari et al.
2007; Czech et al. 2009; Ghildiyal et al. 2010; Okamura
et al. 2009), the 5⬘ nucleotide identity (Chen 2009; Czech

et al. 2009; Okamura et al. 2009), and factors other thansequence features (Hu et al. 2009).

Non-canonical pathways of miRNA biogenesis
We now have a good understanding of the basic process ofmiRNA biogenesis, and current interest is turning toward123Mol Genet Genomics

variations on the canonical mechanism. The analysis of biogenesis of miRNAs has beneWted from deep-sequencing
technologies. Indeed, the lengths of miRNAs make next
generation sequencing instruments ideal for genome-wide
discovery of miRNA genes (Mardis 2008). The sequencing
of small RNA fragments is followed by an extensive bioinformatics-based characterization that examines the genomic locations and the potential for secondary structureformation, to identify putative miRNAs. This approach has
identiWed several classes of miRNA that bypass a key
step(s) involved in the canonical miRNA biogenesis pathway. The challenge for these studies is to distinguishwhether the identiWed miRNA-like small RNA sequences
are merely non-functional degradation products or bona
Wde miRNAs of functional signiWcance. It is important tobear in mind that the functionality of only a small numberof these non-canonical miRNAs has been demonstrated,mostly using artiWcial reporter constructs. However, this isalso the case for canonical miRNAs.

Drosha-independent pathways
Mirtrons
Approximately 40% of animal miRNAs are encoded in the
introns of protein-coding genes and, therefore, many primiRNA transcripts are simultaneously pre-mRNAs and primiRNAs (Cai et al. 2004; Kim et al. 2009). In Drosophila,
the distribution of intron length has a sharp peak at around60 nt (Lim and Burge 2001), which is conspicuously similar to the size of pre-miRNAs. Examination of deep
sequencing data of small RNAs from Drosophila hasrevealed small RNAs that originate from pre-miRNA-sized
short introns with hairpin potential (Okamura et al. 2007;
Ruby et al. 2007). This class of short introns, termed “mirtrons”, can be spliced and debranched into pre-miRNAhairpins that are suitable for Dicer cleavage, thus bypassing
the Microprocessor (Fig. 1b). The mirtron biogenesis pathway converges in the nucleus with the canonical miRNA
pathway during hairpin export, which is mediated byExportin-5 (Okamura et al. 2007) (Fig. 1b). Thus, mirtronmaturation is initiated by splicing and intron lariat debranching, which contrasts with the splicing-independent biogenesis of canonical miRNAs found within introns, whosecleavage by Drosha occurs prior to host intron splicing(Kim and Kim 2007).
In vertebrates, the majority of introns extend to several
kilobases or even megabases in length. However, a considerable fraction of vertebrate introns have a minimal size
(50–150 nt), with the peak at the lower end of the size distribution (Yu et al. 2002). Some of these short introns are
predicted to form hairpin structures, and miRNA-sized

123

RNAs corresponding to these introns are present in collections of mammalian small RNAs (Berezikov et al. 2007;
Babiarz et al. 2008). Thus, mirtrons comprise a small fraction of miRNAs in mammals as well as in Drosophila.
miRNAs derived from snoRNAs
Some small nucleolar RNAs (snoRNAs) also provide a secondary source of pre-miRNAs that is independent of theMicroprocessor-mediated processing (Ender et al. 2008;
Saraiya and Wang 2008) (Fig. 1c). snoRNAs are evolutionarily conserved small RNAs approximately 70–200 nt
long that function as small nucleolar ribonucleoprotein(snoRNP) complexes to guide the enzymatic modiWcationof selected ribosomal RNA (rRNA) nucleotides (Matera
et al. 2007). Analysis of deep sequencing data of small
RNAs associated with immunopuriWed human Ago1 andAgo2 identiWed miRNA-like molecules originating fromparticular snoRNAs, whose secondary structure is characterized by two pre-miRNA-like hairpins linked by a hinge(Ender et al. 2008). Processing of these snoRNAs into miRNAs requires Dicer activity but is independent of Drosha.
These snoRNAs represent functional snoRNAs, because
they associate with speciWc snoRNP proteins such as
GAR1. Thus, some snoRNAs are both the source of miRNAs and the component of snoRNPs that modiWes rRNAs.
Similar snoRNA-derived miRNAs have been identiWed in
Giardia lamblia, which is a unicellular protozoan (Saraiya
and Wang 2008).
These Wndings raise questions as to how snoRNPs normally present in the nucleolus are exported to the cytoplasm for Dicer processing, and how in the cytoplasm
Dicer is recruited onto snoRNAs that are packed with several speciWc proteins. Finally, the identiWcation of miRNAs
generated from snoRNAs in both humans and the unicellular eukaryote Giardia and likely many other organisms,
suggests that snoRNAs may have an ancient link with miRNAs (Scott et al. 2009; Taft et al. 2009).
miRNAs derived from endogenous short-hairpin RNAs
(endo-shRNAs) and tRNA precursors
The analysis of miRNA biogenesis has beneWted from classic mutation analyses combined with deep sequencing.
Mouse embryonic stem cells (mESCs) that lack Dgcr8, thepartner of Drosha in the Microprocessor, have less severe
phenotypes than mESCs lacking Dicer, suggesting a physiological role for Microprocessor-independent, Dicerdependent small RNAs (Babiarz et al. 2008). Analyses ofsmall RNA populations identiWed from wild-type, dgcr8
and dicer knockout mESCs uncovered a diverse populationof Dgcr8-independent, Dicer-dependent small RNAs.
These included miRNAs from mirtrons. In addition, aMol Genet Genomics

fraction of miRNA-like molecules was derived from endoshRNAs.
One of the identiWed endo-shRNAs gives rise to a transcript that forms a secondary structure with two potential
pre-miRNA-like hairpins linked by a hinge (Babiarz et al.
2008). However, only the second hairpin produces a substantial number of sequenced small RNAs (mir-1980)(Fig. 1d). This is reminiscent of snoRNAs producing miRNAs (Ender et al. 2008). The Microprocessor independence
in both cases suggests that an unknown nuclease removes
the Wrst hairpin up to the 5⬘-end, or cleaves at the 5⬘-end of
the second hairpin to produce a suitable Dicer substrate.
Other miRNAs including mir-320 and mir-484, also originate from endo-shRNAs. However, these appear to be transcribed directly as short hairpins.
An endo-shRNA locus corresponds to the isoleucinetRNA (tRNA-Ile) gene (Babiarz et al. 2008). Like othertRNA genes, this locus is transcribed by RNA polymerase
III (pol III) and the transcription is terminated in a poly-U
stretch. Unlike other tRNAs, which typically form cloverleaf secondary structures, the primary transcript from thisgene has the potential to form a long 110 nt hairpin as analternative secondary structure (Fig. 1e). The cluster of
Dgcr8-independent, and Dicer-dependent small RNAs
mapped to the 3⬘-end of the hairpin. The primary transcript
is also processed into a mature tRNA. Thus, this transcript
is a dual function molecule with both miRNA and tRNAprecursors, as is the case for snoRNAs that also give rise tomiRNAs.
tRNase Z-dependent pathways
miRNAs encoded by murine -herpesvirus 68 (MHV68)
are transcribed by pol III to give rise to a pri-miRNA consisting of a tRNA linked to a pre-miRNA hairpin (PfeVer
et al. 2004, 2005; Bogerd et al. 2010). All nine miRNAs
encoded by MHV68 are derived from one arm of one oftwo proposed pre-miRNA-like stem loops located 3⬘ oftRNA-like structures (Bogerd et al. 2010). The pri-miRNAs
are not processed by Drosha but instead by the cellular
tRNA processing enzyme tRNase Z (a.k.a ELAC and 3⬘
tRNase), which cleaves 3⬘ of the tRNA to liberate premiRNA hairpins that are then processed by Dicer to yield
the mature viral miRNAs (Fig. 1f). Therefore, the biogenesis of MHV68 miRNAs is Drosha-independent but isdependent on the action of both a distinct endonuclease,i.e., tRNase Z, and Dicer. Therefore, the MHV68 miRNAs
are distinct from other Drosha-independent miRNAs andare also distinct from other tRNA-derived small RNAs (seebelow). tRNase Z is thought to function in the nucleus(Lund and Dahlberg 1998); thus, this biogenesis pathway
likely converges in the nucleus with the canonical miRNA
pathway during hairpin export.

miRNAs derived from a terminal hairpinof an endo-siRNA precursor
Endogenous siRNAs (endo-siRNAs or esiRNAs) aresmall RNAs derived from long endogenous dsRNAs. In
Drosophila, endo-siRNAs derive from transposons, heterochromatic sequences, intergenic regions, long RNA transcripts with extensive structure and, in some cases, frommRNAs (Okamura and Lai 2008). Although miRNAs areprocessed by Dicer1 and are loaded onto AGO1, endo-siRNAs are generated by Dicer2 and are loaded onto AGO2
(Siomi and Siomi 2009). However, many endo-siRNAs
depend on Loqs, the canonical partner of Dicer1 in themiRNA pathway, but not on R2D2, the partner of Dicer2.
Recent studies have shown that Dicer1 associates with
Loqs isoforms, Loqs-PA and -PB, whereas Dicer2 interacts
with another isoform, Loqs-PD. The production of endosiRNAs strongly depends on Loqs-PD (Hartig et al. 2009;
Miyoshi et al. 2010; Zhou et al. 2009). These diVerent Dicer
complexes recognize the stem length of precursor hairpins(Miyoshi et al. 2010). This suggests that, besides the intrinsic substrate speciWcity of the individual Dicer proteins, theidentity of Dicer-interacting cofactors is also crucial for thebiogenesis of diVerent classes of small RNAs.
A subset of endo-siRNAs is derived from long RNA
hairpins containing multiple mismatches, which are processed to mature endo-siRNAs by the Dicer2-Loqs-PD
complex. Although mature endo-siRNAs are loaded onto
AGO2, small RNAs derived from the pre-miRNA-sized
terminal hairpin of the endo-siRNA precursor were foundassociated with AGO1. This result suggests that the Dicer
2-Loqs-PD complex processes the precursors to produce
endo-siRNAs but cannot process the terminal hairpins,
which instead are recognized as miRNA precursors andprocessed to miRNAs by the Dicer1-Loqs-PA/PB complex(Miyoshi et al. 2010) (Fig. 1g). These miRNAs are aunique subset of miRNAs, and are referred to as ‘siblingsof esiRNAs’ or semiRNAs.

Dicer-independent pathways
Argonaute proteins are the core components of RISCs thatmodulate gene expression, regulate chromosome structureand function and provide an innate immune defense against
viruses and transposons (Hutvagner and Simard 2008).
AGO proteins consist of an amino-terminal domain, theMid domain and their signature PAZ and PIWI domains(Parker 2010). The PAZ and Mid domains help to anchor
the small RNA guide, with PAZ binding the 3⬘-end of the
RNA and the Mid domain providing a binding pocket forthe phosphorylated 5⬘-end. The PIWI domain forms an
RNase H-like fold in the tertiary structure (Song et al. 2004).

123

Mol Genet Genomics

Loading of a highly complementary target into an AGO
protein brings the scissile phosphate, opposite nucleotides
10 and 11 of the small RNA guide, into the endonuclease(Slicer) active site, which consists of a catalytic DDH triad
that serves as a metal-coordinating site (Elbashir et al.
2001; Liu et al. 2004; Schwarz et al. 2004; Song et al.
2004). Of four AGO proteins in mammals, only Ago2 hasretained both the DDH motif and demonstrable Slicer
activity (Azuma-Mukai et al. 2008; Liu et al. 2004; Meister
et al. 2004).
The Hannon group has produced mice with a catalytically inactive Ago2 that disrupts RNA cleavage without
impeding small RNA binding (ChelouW et al. 2010). Micelacking catalytic Ago2 died shortly after birth with anemia.
Ago2 catalytic activity is important for postnatal development. Deep sequencing of miRNAs in the liver of the Ago2
mutants revealed that miR-451 was markedly reduced in
the mutants, although other miRNAs were present at nearlynormal levels. The miR-451 precursor has an unusual feature: although canonical miRNAs are deWned as mapping to
the stem only, the six terminal nucleotides of the 23-nt long
mature miR-451 span the loop region and extend into thecomplementary strand of the hairpin precursor. In drosha¡/¡mouse embryonic Wbroblasts, mature miR-451 was reduced
by 20-fold compared with wild-type levels, and in vitro
processing assays using puriWed Drosha complexes with
miR-451 primary transcripts revealed that the substrate wasprocessed to a pre-miRNA only 41-nt long, which is also
unusual. These experiments provide both genetic andbiochemical support for Drosha catalyzing the excision ofpre-miR-451 from its primary transcript. The levels of
miR-451 were unaVected in dicer null cells, indicating thatthe conversion of pre-miR-451 into a mature miRNAproceeds independently of Dicer. Thus, the maturationof miR-451 is Drosha-dependent and Dicer-independent(Fig. 1h).
Pre-miR-451 was detected in Ago2 immunoprecipitates.
In processing assays using aYnity-puriWed Ago2 and premiR-451, mature miR-451 was produced depending on
Ago2 catalytic activity. Together these Wndings suggest thatpre-miR451 produced by Drosha is directly loaded onto
Ago2 without the Dicer step, and the Ago2 catalytic centre
helps to catalyze the maturation of this miRNA (Fig. 1h).
This is at odds with earlier reports indicating a coupling ofDicer cleavage and RISC loading (Chendrimada et al.
2005; Wang et al. 2009). However, the miR-451 pathway isconserved in zebraWsh (Cifuentes et al. 2010). miR-451 has
a deWned 5⬘-end created by a Drosha cut, but a variable 3⬘end that extends over the loop region and ranges between
20 and 30 nt long. Reads stopped at nucleotide 30 measured
from the 5⬘-end of the pre-miRNA, which is the Ago2
cleavage site, and longer reads contained 1–5 non-templated uridines. This suggests a model whereby Ago2-med-123

iated cleavage produces an intermediate that is uridylated
and is then further trimmed by a cellular nuclease to generate mature miRNA sequences protected by Ago2 (Fig. 1h).
These Wndings also link the conservation of Ago2’s Slicer
activity to a conserved mechanism of miRNA biogenesis
and suggest the possibility that additional miRNAs may
well rely on the Ago2-dependent pathway for maturation.
Several studies have identiWed a large number ofmiRNA-sized small RNAs derived from human tRNAs(Cole et al. 2009; Kawaji et al. 2008; Lee et al. 2009;
Haussecker et al. 2010). At least a fraction of these tRNAderived small RNAs is associated with Argonaute proteins(Cole et al. 2009; Haussecker et al. 2010). These tRNAderived small RNAs are largely subdivided into twogroups: Dicer-dependent and Dicer-independent but tRNase Z-dependent. Pre-tRNAs produced by pol III are
trimmed by RNase P at the 5⬘-end (Altman 2000) and bytRNase Z at the 3⬘-end (Weiner 2005). The phosphorylated
5⬘-ends of these tRNA-derived small RNAs are generated
from pre-tRNAs by RNaseZ cleavage (Mayer et al. 2000),
while the 3⬘-end results from transcription termination by
pol III. Those derived in a Dicer-independent and tRNase
Z-dependent manner are 3⬘-trailer sequences from pretRNAs, released by a cleavage by tRNase Z (Fig. 1i). They
do not even partially meet the classical deWnition of amiRNA and are, therefore, called either tRNA-derived
RNA fragments (tRFs) (Lee et al. 2009) or type II tRNAderived small RNAs (tsRNAs; type I tsRNAs being Dicerdependent) (Haussecker et al. 2010). Because the 3⬘-end ofalmost all eukaryotic RNAs including polyadenylated
mRNAs and non-polyadenylated histone mRNAs are generated by speciWc nucleolytic cleavage (Weiner 2005), 3⬘trailer sequences from these precursors could be anothersource of small RNAs. Although the precise function ofthese tRNA-derived small RNAs is not known, it is tempting to speculate that tRNAs, as an abundant class of RNAs,
compete with pre-miRNAs for Dicer, and tRNA-derived
small RNAs compete with miRNAs for Argonaute proteins,thereby regulating the abundance of miRISCs and maintaining miRNA homeostasis.

Conclusions
Cells take advantage of a wide variety of mechanisms togenerate miRNAs and miRNA-like molecules, and have
evolved the means to use a variety of transcripts as sourcesof miRNAs. Non-canonical pathways of miRNA biogenesis demonstrate the ability and Xexibility of cells to generate pre-miRNA-like hairpins that are routed to Dicer and/or
5⬘-phosphorylated small RNAs that bind directly to AGOs.
On the other hand, current data also raise the possibility
that any cellular RNAs with potential pre-miRNA-likeMol Genet Genomics

hairpins could be substrates for Dicer. Thus, there must be
systems in eukaryotic cells to protect transcripts withpotential hairpins against Dicer and/or regulate their availability to Dicer.
There are many future challenges. How many classes of
non-canonical miRNAs exist? How are these miRNAs generated? How is the processing of these non-canonical miRNAs used to regulate the activity of canonical miRNAs?
What are their biological functions? Some snoRNAs andtRNAs are dual function molecules with both miRNA andsnoRNA or tRNA capabilities. This already hints that otherabundant non-coding RNAs such as rRNAs and U snRNAs
among others (Hüttenhofer and Schattner 2006) may wellprovide sources of non-canonical miRNAs.
Acknowledgments The authors would like to thank the members of
the Siomi Laboratory for discussions. This work was supported by
MEXT grants to H.S and K.M, and a Keio University Grant-in-Aid for
Encouragement of Young Medical Scientists to K.M.

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