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First published online 20 February 2008
doi: 10.1242/dev.005629
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Review |
1 Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The
Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10065,
USA.
2 Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, 1275 York
Avenue, New York, NY 10065, USA.
Author for correspondence (e-mail:
ttuschl{at}rockefeller.edu)
SUMMARY
Several distinct classes of small RNAs, some newly identified, have been discovered to play important regulatory roles in diverse cellular processes. These classes include siRNAs, miRNAs, rasiRNAs and piRNAs. Each class binds to distinct members of the Argonaute/Piwi protein family to form ribonucleoprotein complexes that recognize partially, or nearly perfect, complementary nucleic acid targets, and that mediate a variety of regulatory processes, including transcriptional and post-transcriptional gene silencing. Based on the known relationship of Argonaute/Piwi proteins with distinct classes of small RNAs, we can now predict how many new classes of small RNAs or silencing processes remain to be discovered.
Introduction
Small RNAs perform diverse biological functions, often in a tissue-specific
manner. They function by guiding sequence-specific gene silencing at the
transcriptional and/or post-transcriptional level (reviewed by
Bartel, 2004
;
Meister et al., 2004;
Nakayashiki, 2005;
Vaucheret, 2006;
Grewal and Jia, 2007;
Seto et al., 2007;
Zaratiegui et al., 2007).
Naturally occurring small RNAs are processed from longer RNA precursors that
are either encoded in the genome or are generated by viral replication.
Importantly, these natural RNA-silencing processes can be harnessed to induce
gene-specific silencing through the provision of non-natural RNA precursors or
mimics of natural, small RNA processing intermediates. This approach, known as
RNA interference (RNAi), is widely used for the systematic analysis of gene
function, and its potential therapeutic applications are currently under
intense investigation (reviewed by Bumcrot
et al., 2006; Echeverri and
Perrimon, 2006). However, to efficiently harness the machinery of
RNAi, it is essential to elucidate how the different types of small RNA
molecules are generated.
Distinct sequence and/or structural elements within the precursor
transcripts of various classes of small RNAs recruit RNA-processing enzymes
and proteins that are responsible for small RNA maturation, and also for the
subsequent assembly of the small RNAs into effector complexes that mediate
small RNA function. The best-characterized RNA structure that triggers RNAi is
double-stranded RNA (dsRNA), either in the form of a hairpin (>20 bp) or a
longer dsRNA. Recently, small RNA classes that may originate from apparently
single-stranded RNA (ssRNA) transcripts have also been identified
(Aravin et al., 2006;
Girard et al., 2006;
Grivna et al., 2006a;
Lau et al., 2006;
Ruby et al., 2006;
Brennecke et al., 2007). The
key feature that distinguishes the different classes of small RNAs from each
other is their length, with peak lengths varying from 21 to 30 nucleotides
(nt). The lengths of the different classes of small RNAs vary due to distinct
mechanisms of biogenesis. Other significant differences between them are the
presence of a 5' uridine, phosphorylation at the 5' end, and
2'-O-methylation at the 3' end of the RNA molecule.
These characteristics of small RNAs determine their loading onto effector
ribonucleoprotein (RNP) complexes. These effector complexes mediate different
small RNA functions at the transcriptional and/or post-transcriptional level,
such as mRNA cleavage, translational repression, and regulation of chromatin
structure. For example, the effector complex that mediates catalytic mRNA
cleavage is known as RNA-induced silencing complex (RISC), the effector
complex that mediates translational repression directed by microRNAs (miRNAs)
is known as miRNP, and the effector complex that mediates chromatin regulation
is the RNA-induced transcriptional gene silencing (RITS) complex (reviewed by
Meister and Tuschl, 2004).
Small RNA-associated RNP complexes contain at their center an
Argonaute/Piwi (Ago/Piwi) protein family member (see
Table 1) and are loaded with
distinct classes of small RNAs to form target-recognizing complexes
(Hammond et al., 2001;
Hutvagner and Zamore, 2002;
Martinez et al., 2002;
Mourelatos et al., 2002;
Meister et al., 2004;
Aravin et al., 2006;
Grivna et al., 2006b;
Lau et al., 2006;
Saito et al., 2006;
Vagin et al., 2006;
Watanabe et al., 2006;
Brennecke et al., 2007;
Gunawardane et al., 2007;
Houwing et al., 2007). The
number of Ago/Piwi genes varies considerably among species, setting an upper
limit on the number of classes of small regulatory RNAs that remain to be
identified and the number of small RNA-guided regulatory processes. The tissue
specificity that is associated with the expression of various members of the
Ago/Piwi protein family and their small RNA precursors add further complexity
to our understanding of small RNA-regulated processes.
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The Ago/Piwi protein family
The Ago/Piwi protein family is well conserved, and members have been
identified in all species that possess small RNA-mediated phenomena (see
Fig. 1A) (reviewed by
Parker and Barford, 2006;
Peters and Meister, 2007).
Based on their sequence similarities, Ago/Piwi proteins can be divided
phylogenetically into three families (see
Fig. 1A). The largest family
comprises the Argonautes (Ago), named after its founding member in
Arabidopsis thaliana. The second family comprises the Piwis, named
after the Drosophila melanogaster protein PIWI (P-element induced
wimpy testis). The third family, Class 3, consists exclusively of
Caenorhabditis elegans proteins. Different members of the Ago/Piwi
family often show distinct tissue distribution, which allows some to mediate
tissue-specific small RNA functions (see
Table 1). The importance of
individual members of the Ago/Piwi protein family has been assessed in many
genetic studies (see Table 1).
These studies, however, are sometimes complicated by redundant or overlapping
Ago/Piwi protein functions and expression.
Ago/Piwi proteins have a molecular weight of 
90 kDa and show an
overall bilobal architecture (see Fig.
1B). The first lobe contains the N-terminal PAZ-domain that is
responsible for binding the 3'-end of the guide small RNA. The second
lobe contains the MID-domain, responsible for binding the 5'-phosphate
of the guide RNA, and the RNase H endonuclease domain, also known as the
PIWI-domain (reviewed by Parker and
Barford, 2006; Patel et al.,
2006; Tolia and Joshua-Tor,
2007).
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). Most
miRNAs and small interfering RNAs (siRNAs) are initially bound to the
Ago/Piwi-containing RNP complex as duplexes with a two nt 3' overhang,
and are subsequently unwound, resulting in bound ssRNA intermediates. The
bound ssRNA strand is referred to as the `guide' RNA, whereas the ssRNA strand
not stably incorporated into the RNP complex is referred to as the `passenger'
or `star' RNA, and is cleaved by the Ago/Piwi proteins
(Matranga et al., 2005;
Miyoshi et al., 2005). A PAZ
domain is also found in most Dicer RNase III family members, where it is
believed to position one end of the RNA duplex at a defined distance from the
endonuclease domain, thereby producing small RNAs of a defined length
(Zhang et al., 2004;
Macrae et al., 2006;
MacRae et al., 2007). Dicer
RNase III family members are endonucleases required for the biogenesis of
specific classes of small RNAs by generating small dsRNAs from longer dsRNA
precursors (see more detailed discussion below).
The MID domain has structural homology to the sugar-binding domain of the
lac repressor and is around 150 amino acids. It loads the small RNA
onto the RNP complex (Nykanen et al.,
2001), presumably by receiving and binding the 5' phosphate
of the small RNA presented as a duplex
(Chen et al., 2007). It places
the 5' phosphate end of a small RNA in a binding site that is formed by
a basic pocket of the MID domain adjacent to the interface with the PIWI
domain, the C-terminal carboxylate of the Ago/Piwi protein and a divalent
metal cation, such as magnesium (Ma et
al., 2005; Parker et al.,
2005; Rivas et al.,
2005). Within the RISC RNP, the presence of a 5' phosphate
on the bound single-stranded siRNA contributes to the fidelity of the
endonuclease activity during target cleavage
(Rivas et al., 2005). The MID
domain of some Ago proteins contains a sequence motif similar to the
methyl(7)G-cap-binding domain of the eukaryotic translation initiation factor
eIF4E. The ability of Ago/Piwi proteins to bind the m(7)G cap of the target
mRNA suggests that one mechanism of small RNA regulation occurs by controlling
the translation initiation of their target mRNAs
(Kiriakidou et al., 2007).
The PIWI domain exhibits a RNase H fold, and ranges between 400-600 amino
acids. The RNase H domain is conserved among eukaryotes and prokaryotes. It
may act as a double-strand-specific endonuclease (also referred to as
`Slicer') that can cleave the mRNA targeted by the guide small RNA. Ago/Piwi
protein members show sequence variation in the active site, and not all
members have endonuclease activity (see
Table 1). Some Ago/Piwi
proteins include the Asp-Asp-His motif that forms the active site catalytic
triad, possessing endonuclease activity with a divalent cation, such as
calcium (reviewed by Tolia and
Joshua-Tor, 2007). A recent study has revealed that the PIWI
domain binds a conserved motif in Ago/Piwi interacting proteins, such as GW182
and Tas3. These proteins are involved in mediating the small RNA-Ago/Piwi
complex functions in the processes of translational repression and
transcriptional silencing, respectively (see subsequent sections)
(Till et al., 2007).
Small RNAs: their biogenesis and function
An overview of the classes of small RNAs and their Ago/Piwi protein-binding partner is shown in Table 1. The molecular characteristics of the small RNAs, including their length, precursor structure and chemical modifications are presented in Table 2. It is sometimes difficult to draw a clear line between the different classes of small RNAs, partly because the nomenclature that was introduced early on in the field did not anticipate the complexity of the small RNAs and the many processes they mediate. The features that can be used to distinguish different classes of small RNAs are: mechanism of biogenesis (or precursor structure); the genomic region they originate from; and their associated protein-binding partner. Classes of small RNAs can be grouped into two main categories, those excised from dsRNA precursors and those derived from transcripts that are probably not double stranded. The best-characterized members of the first category are siRNAs and miRNAs, whereas members of the second category include Piwi-interacting small RNAs (piRNAs) and some repeat-associated-siRNAs (rasiRNAs). In this section, we describe the different classes of small RNAs that have been identified to date and their functions, and in the next section we describe in more detail the proteins involved in their biogenesis.
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miRNAs are the most abundant class of small RNAs in animals. They are on
average 20 to 23 nts in length and usually have a uridine at their 5'
end. The first representative of this small RNA family, lin-4, was
identified in a genetic screen in C. elegans in 1981
(Chalfie et al., 1981), and
was molecularly characterized in 1993 (Lee
et al., 1993). Plants have on average 120 miRNA-encoding genes
(reviewed by Jones-Rhoades et al.,
2006), invertebrate animals about 150
(Aravin et al., 2003;
Lai et al., 2003;
Ruby et al., 2006), and
humans close to 500 (Landgraf et al.,
2007), which are differentially expressed depending on the cell
type and developmental stage. miRNAs have also been identified in DNA viruses
(Pfeffer et al., 2004;
Pfeffer and Voinnet, 2006)
and the green algae Chlamydomonas reinhardtii
(Molnar et al., 2007;
Zhao et al., 2007). miRNAs
can be expressed at high levels, up to ten thousands of copies per cell, and
can thus play important regulatory roles by controlling hundreds of mRNA
targets (Lim et al., 2003). A
repository of miRNAs and miRNA genes from many organisms is available at the
miRBase Sequence Database
(http://microrna.sanger.ac.uk/sequences/),
a searchable database of published miRNA sequences and annotation. An
expression atlas/database of mammalian miRNAs identified in a variety of
tissues and cell lines has also recently become available
(www.mirz.unibas.ch/smiRNAdb/).
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). In animals, they are processed by endoribonucleases in
partnership with dsRNA-binding proteins sequentially in the nucleus and
cytoplasm (see Fig. 2). In the
animal nucleus, the endoribonuclease Drosha excises the miRNA stem loops from
the primary transcript (pri-miRNA), producing an approximately 70 nt
intermediate (pre-miRNA) (Lee et al.,
2002). The pre-miRNA is actively transported to the cytoplasm in a
GTP-dependent manner by an export protein complex containing a dsRNA-binding
export receptor, such as mammalian Exportin 5 or plant HASTY, and Ran
(ras-related nuclear protein) GTPase (reviewed by
Kim, 2004). In the cytoplasm,
the pre-miRNA is further processed by Dicer to the mature miRNA in the form of
a base-paired double-stranded processing intermediate with a 2 nt 3'
overhang. In plants, nuclear-localized Dicer is responsible for pri-miRNA
processing to miRNA, followed by the addition of a 2'-O-methyl group at
the 3' end of the miRNA by a methyltransferase (reviewed by
Vazquez, 2006). In the
cytoplasm, the strand of the duplex whose 5' end is less stably paired
is favored for incorporation into Ago effector complexes
(Schwarz et al., 2003). The
other strand, referred to as miRNA*, becomes degraded.
An alternative miRNA biogenesis mechanism has recently been identified, the
precursors of which reside in introns. In these intronic miRNAs, named
mirtrons, the 3' end of the stem-loop precursor structure coincides with
the 3' splice site, and is cleaved by nuclear pre-mRNA splicing rather
than by Drosha (Berezikov et al.,
2007; Okamura et al.,
2007; Ruby et al.,
2007).
miRNA function and Ago/Piwi association
miRNAs have been implicated in many cellular processes by regulating gene
expression at the post-transcriptional level. miRNA RNPs mediate diverse
functions depending on the particular Ago protein member and the degree of
sequence complementarity between the guide miRNA and the target nucleic acid
(reviewed by Eulalio et al.,
2007; Peters and Meister,
2007; Pillai et al.,
2007). Several lines of evidence have identified that the six to
eight nts at the 5' end of an miRNA (position 1-8) are important for
target site recognition and have been designated the `seed' region
(Lai et al., 2003;
Lewis et al., 2003;
Stark et al., 2003;
Jackson et al., 2006;
Lall et al., 2006) (reviewed
by Rajewsky, 2006;
Sood et al., 2006;
Gaidatzis et al., 2007).
miRNA RNP effector complexes guide catalytic target RNA cleavage based on
Ago protein sequence variation (see Table
1) at or near the active site, as well as on the degree of
mismatches between the miRNA and target RNA
(Jackson et al., 2003;
Martinez and Tuschl, 2004;
Jackson et al., 2006). Most
miRNA RNPs with near-perfect complementary guide miRNA/target mRNA mediate
mRNA cleavage, whereas RNPs with a greater degree of mismatches inhibit
translation and/or trigger the transport of mRNA to mRNA-processing bodies
(P-bodies, also known as cytoplasmic GW-bodies) (reviewed by
Zamore and Haley, 2005;
Valencia-Sanchez et al.,
2006; Du and Zamore,
2007; Parker and Sheth,
2007; Pillai et al.,
2007). The presence of P-bodies is now considered to be a
consequence of small RNA-guided mRNA targeting
(Eulalio et al., 2007;
Lian et al., 2007). Components
of the RNAi machinery localized to P-bodies include members of the Ago/Piwi
family, members of the GW-proteins/trinucleotide repeat-containing family of
proteins, and RNA helicases (reviewed by
Ding and Han, 2007;
Parker and Sheth, 2007).
Other proteins concentrated in P-bodies include general mRNA translation
repression and mRNA decay machinery proteins, such as mRNA decapping proteins,
translational repressors, deadenylase complexes and RNA-binding proteins.
Small RNA-mediated regulation does not necessarily require localization to
P-bodies, and P-bodies are not always detectable (reviewed by
Eulalio et al., 2007;
Jakymiw et al., 2007).
Interestingly, GW-proteins are also involved in the crosstalk between the
maternal macronucleus and the developing macronuclei during RNA-mediated DNA
elimination processes in the ciliate Paramecium tetraurelia
(Nowacki et al., 2005)
following the sexual process of conjugation.
In many organisms there is biochemical evidence that miRNAs specifically
associate with members of the Ago family. All four human AGO proteins
(Liu, J. et al., 2004;
Meister et al., 2004), A.
thaliana AGO1 (Vaucheret et al.,
2004; Baumberger and Baulcombe,
2005; Qi et al.,
2005), and C. elegans Alg-1 and Alg-2
(Grishok et al., 2001)
interact with miRNAs. Certain members of the miRNA-associated Ago proteins
exhibit endoribonuclease activity and are thus capable of target mRNA cleavage
(see Table 1). More recently,
it has become apparent that D. melanogaster Ago1 preferentially binds
miRNAs that have been excised from imperfectly paired hairpin precursors,
whereas those miRNAs that have near-perfectly paired hairpin precursors are
bound by Ago2 (Okamura et al.,
2004; Miyoshi et al.,
2005; Forstemann et al.,
2007; Tomari et al.,
2007).
miRNA conservation
Many miRNAs are represented as families that are defined by the
conservation of the seed region. miRNAs identified in one species are often
conserved in closely related species (see miRBase), and about 10% of the miRNA
families identified in invertebrates are completely conserved in mammals.
There is no sequence conservation between the miRNAs of animals and plants.
Plant and animal miRNAs have different 3'-end modifications: plant
miRNAs are 2'-O-methylated (Yu et
al., 2005), whereas animal miRNAs are unmodified
(Kirino and Mourelatos, 2007b;
Ohara et al., 2007). Animal
and plant miRNAs also have different mRNA target-recognition modes: plant
miRNAs usually cleave in open reading frames (ORFs), whereas the binding sites
of animal miRNAs are most often located in 3' untranslated regions
(UTRs) (reviewed by Bartel,
2004; Stark et al.,
2005; Gaidatzis et al.,
2007). Moreover, plant miRNAs show a greater degree of
complementarity to their mRNA target than do animal miRNAs, and primarily
function through mRNA cleavage. Animal miRNAs target mRNA 3' UTRs
predominantly by seed sequence complementarity and are rarely fully
complementary; they therefore function through translational repression rather
than cleavage. A recent study in mammals revealed that the sequence that
surrounds the 3' UTR target region that is complementary to the miRNA
seed region also contributes to the repression of a target mRNA by a miRNA
(Grimson et al., 2007;
Nielsen et al., 2007).
siRNAs
The first hunch that small RNAs mediate gene silencing came from their
observation in transgenic co-suppressing plants
(Hamilton and Baulcombe,
1999). Co-suppression is triggered by the genomic integration of
an additional gene (or of gene segments) that is identical to a host gene, and
results in the reduced accumulation of RNA molecules that share sequence
similarity with the introduced nucleic acid. Biochemical studies following the
discovery of RNAi in C. elegans
(Fire et al., 1998) revealed
that small RNAs were processed from dsRNA triggers
(Zamore et al., 2000).
Because these dsRNA processing products were able to efficiently reconstitute
silencing complexes, they were named siRNAs
(Elbashir et al., 2001a).
siRNA biogenesis
siRNAs have a distinct size distribution, but, in contrast to miRNAs, which
are excised in a precise fashion from their dsRNA precursor, siRNAs are
processed in a more random fashion
(Elbashir et al., 2001a) from
longer dsRNAs (see Fig. 3A)
(Hammond et al., 2000;
Zamore et al., 2000). They
are processed by Dicer, producing two nt 3' overhangs, similar to the
final processing intermediate of the miRNA pathway.
siRNAs can be produced from RNA transcribed in the nucleus (endogenous
siRNAs), or can be virally derived or experimentally introduced as chemically
synthesized dsRNA (exogenous siRNAs). Endogenous plant siRNAs can be generated
directly from transcription or can be derived from inverted repeats of
transgenes or transposons. They include natural antisense-siRNAs (natsiRNAs),
trans-acting-siRNAs (tasiRNAs) and heterochromatic small RNAs (hcRNAs).
natsiRNAs are endogenously expressed siRNAs that originate from overlapping
sense and antisense transcripts (Borsani
et al., 2005). tasiRNAs are generated from specific non-coding
genomic regions. Their biogenesis is initiated by Ago1-bound miRNAs that
cleave the non-coding ssRNA transcript to produce fragments, which serve as
templates for dsRNA synthesis by a RNA-dependent RNA polymerase (RdRP, RDR6)
(see Fig. 3A). The dsRNA
fragment is subsequently cleaved by a Dicer RNase (Dicer-like 4, DCL4) to
yield 21 nt tasiRNAs (reviewed by
Vazquez, 2006). Plant hcRNAs
are mostly derived from repeat-associated genomic regions (see below).
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; Simmer et al.,
2003; Ruby et al.,
2006). They include the previously annotated tiny-noncoding RNA
(tncRNA) and secondary siRNA. tncRNAs are 22 nts in length, depend on
Dicer for their biogenesis, and derive from non-coding, non-conserved
sequences. If RNAi is induced in C. elegans, primary siRNAs derived
from the processing of the trigger dsRNA are generated, as are secondary
siRNAs that originate from the unprimed RdRP synthesis of dsRNA (see
Fig. 3A)
(Pak and Fire, 2007;
Sijen et al., 2007). These
siRNAs are 21 to 22 nts in length, are of antisense polarity to the targeted
gene, and have 5' di- or triphosphate termini.
So far, endogenous siRNAs have not been identified in mammals or insects.
In cultured mammalian cells, siRNAs have been successfully used to analyze
gene function (Elbashir et al.,
2001b). The exposure of mammalian cells to long dsRNA induces an
antiviral interferon response that leads to apoptosis (reviewed by
Dorsett and Tuschl, 2004).
This reaction can be bypassed by using siRNA duplexes that resemble in size
and structure the miRNA processing intermediates. In this setting, siRNAs
depend on the cellular miRNA machinery for their function and guide the
cleavage of target RNA by binding to Ago2
(Liu, J. et al., 2004).
siRNA function and Ago/Piwi association
siRNAs associate with Ago family members to form siRNA RNP complexes (known
as RISC) that guide target mRNA cleavage. Exogenous siRNAs trigger RNAi when
provided as a dsRNA with a two nt 3' overhang, similar to the miRNA or
endogenous siRNA intermediates processed by Dicer. Endogenous siRNAs and RNAi
are thought to play an important role in defending genomes against transgenes
and transposons, as well as against foreign nucleic acids, such as viruses.
The random integration of new DNA or the rearrangement of existing sequences,
such as by transposons, might trigger the formation of dsRNA. dsRNA might also
be generated as a consequence of viral replication or by the action of
genome-encoded RdRPs. As discussed below, another RNAi-related mechanism
involving piRNAs is also involved in genome defense, predominantly in the
germline. Finally, plant and S. pombe hcRNAs play a role in
heterochromatin regulation.
In plants, siRNAs are readily identified from virus- and viroid-infected
cells, or from transgenic plants that show co-suppression (reviewed by
Voinnet, 2005). They fall
mainly into two size classes, 21 to 22 nt and 24 nt species. The shorter
siRNAs (such as tasiRNAs, natsiRNAs, most viral-derived siRNAs) guide mRNA
degradation, while the longer ones (such as hcRNAs) are involved in DNA and
histone methylation (see Fig.
3B). Genetic studies suggest that tasiRNAs may form complexes with
Ago7 that mediate the cleavage of target mRNAs that are different from the
sequences from which the tasiRNAs originate, playing a crucial role in plant
development (Adenot et al.,
2006).
In C. elegans, the function of tncRNAs has not yet been
elucidated. Secondary siRNAs appear to associate with the Class 3 Ago/Piwi
proteins Sago-1 and Sago-2, and their function is to support the primary siRNA
signal (Yigit et al.,
2006).
Small RNAs derived from repetitive genomic sequence
Small RNA sequences identified in clone libraries that do not map to a single genomic region but to many, sometimes thousands of sites, are classified as being repeat derived. Depending on the species and their size distribution, these small RNAs can be classified as being a category of conventional siRNAs (hcRNAs), or as constituting their own class, defined by a distinct mechanism of maturation, and by the Ago/Piwi protein they associate with (rasiRNAs).
hcRNAs
hcRNAs identified in Saccharomyces pombe, plants and
Trypanosoma brucei are siRNAs that derive from long dsRNA precursors
that are transcribed from genomic repeat regions (sometimes also referred to
as rasiRNAs). They were initially termed small heterochromatic siRNAs
(shRNAs). However, the abbreviation `shRNA' can be misleading, as it was also
introduced as an abbreviation for `small hairpin RNA', a precursor for stable
expression of siRNAs used for gene silencing.
In the unicellular eukaryote T. brucei, hcRNAs are involved in
transposon control, whereas in S. pombe and A. thaliana they
are also involved in the regulation of heterochromatin structures, and thus
mediate transcriptional gene regulation
(Djikeng et al., 2001;
Reinhart and Bartel, 2002).
In S. pombe, hcRNAs derive from peri-centromeric and mating-type
locus repeats, and have been identified in an Ago-containing effector complex
(RITS) (see Fig. 3B) (reviewed
by Grewal and Jia, 2007). The
RITS complex, in addition to Ago, consists of Tas3 (targeting complex subunit
3), an S. pombe-specific protein, and Chp1 (chromodomain protein 1),
a chromodomain containing protein. The RITS complex subsequently pairs with
the nascent transcript repeat sequences, and recruits the RNA-directed RNA
polymerase complex (RDRC) and Clr4 (cryptic loci regulator 4), a histone
methyltransferase (see Fig.
3B). This complex has been implicated in nucleation and/or
maintenance of heterochromatin by targeting transcripts that emerge from
repeat-containing regions and that are supposed to be transcriptionally
repressed, thereby establishing a feedback loop that reinforces and sustains
the transcriptional silencing of heterochromatic regions. In A.
thaliana, the Ago-siRNA complex associates with DRD1 (defective in
RNA-directed DNA methylation 1), an SNF2-like chromatin remodeling protein,
and with Polymerase IVb, to initiate cytosine methylation via DRM2 (domain
rearranged methyltransferase), a DNA methyltransferase. S. pombe
hcRNAs associate with Ago1 (Irvine et al.,
2006; Buker et al.,
2007), whereas A. thaliana hcRNAs interact with Ago4 and
Ago6 (Zilberman et al., 2003;
Zheng et al., 2007).
rasiRNAs
A subset of rasiRNAs was identified by cloning from D.
melanogaster and D. rerio small RNA libraries
(Aravin et al., 2003;
Chen et al., 2005;
Houwing et al., 2007). Given
their association with the Piwi protein family, they are also known as piRNAs
and are discussed in the following section.
piRNAs and rasiRNAs
piRNAs
piRNAs are 28 to 33 nts in length and have been characterized by the
cloning of small RNAs from anti-Piwi immunoprecipitates prepared from
mammalian testes (reviewed by O'Donnell
and Boeke, 2007).
piRNA biogenesis
Mammalian piRNAs are not usually derived from repeat sequences, given that
the proportion of repeat elements able to generate piRNAs is actually smaller
within the piRNA regions than the frequency of repeat sequences in the mouse
genome (12-20% versus 38%) (Betel et al.,
2007). They are believed to be processed from single-stranded
primary transcripts that are transcribed from defined genomic regions and have
a preference for a uridine at their 5' end (see
Fig. 4A). Mammalian piRNAs are
a highly complex mix of sequences, with tens of thousands of distinct piRNAs
generated from the 50 to 100 defined primary transcripts
(Aravin et al., 2006;
Girard et al., 2006;
Grivna et al., 2006b;
Lau et al., 2006;
Watanabe et al., 2006). This
may suggest that mammalian piRNAs, unlike miRNAs, are not processed in a
precise manner. However, approximately 20% of all piRNA sequences were cloned
three or more times, and many piRNA sequences from the same strand are
partially overlapping, suggesting a quasi-random mechanism
(Betel et al., 2007). The
mechanism of biogenesis of D. melanogaster rasiRNAs is beginning to
be elucidated, and may offer parallels for a specific mode of processing for
piRNAs as well (see section on biogenesis of rasiRNAs below). piRNA biogenesis
is thought to be Dicer independent (Vagin
et al., 2006) and they appear to be 2'-O-methylated at their
3' end (Horwich et al.,
2007; Kirino and Mourelatos,
2007b; Kirino and Mourelatos,
2007a; Ohara et al.,
2007; Saito, K. et al.,
2007).
piRNA conservation and function
Between mammals, mature piRNAs are not conserved; however, the genomic
regions, from which they derive, in particular the promoter sequences, are
conserved (Betel et al., 2007).
Mammalian piRNAs are strongly expressed in the male germline, their total
number per cell obtained from testis tissue reaching up to two million, i.e.
about 10-fold higher than the miRNA content of these cells
(Aravin et al., 2006;
Girard et al., 2006;
Grivna et al., 2006a;
Lau et al., 2006). Although
the targets of piRNAs and their mechanism of action are unknown, the knockout
in mice of any of the testis-expressed three Piwi proteins (Mili, Miwi, Miwi2)
abolishes spermatogenesis (reviewed by
O'Donnell and Boeke, 2007;
Klattenhoff and Theurkauf,
2008). Mammalian piRNAs may also play a role in transposon
regulation, but their mechanism of action is currently uncharacterized. The
knockout of the gene that encodes the Piwi protein Miwi2 in mice leads to
phenotypes that may be linked to an inappropriate activation of transposable
elements (Carmell et al.,
2007). Mice mutant for the Piwi protein Mili also show transposon
de-repression, thus suggesting that mammalian piRNAs may contribute in some
manner to the silencing of transposable elements
(Aravin et al., 2007).
|
;
Brennecke et al., 2007;
Gunawardane et al., 2007;
Nishida et al., 2007). In
D. rerio, rasiRNAs associate with the Piwi protein Ziwi
(Houwing et al., 2007). The
rasiRNAs of D. melanogaster and D. rerio are distinct from
conventional 20 to 23 nt siRNAs or miRNAs, their length ranging between 23 and
28 nt. They have a bias for a uridine at their 5' end, and are
2'-O-methylated at their 3' end
(Houwing et al., 2007;
Saito, T. et al., 2007).
Based on genome content, more D. melanogaster and D. rerio
rasiRNAs than expected originate from retrotransposon sequences, and from
certain repeat-rich genome regions (Betel
et al., 2007). rasiRNAs play a crucial role in controlling the
expression of homologous sequences dispersed throughout the genome (reviewed
by O'Donnell and Boeke,
2007).
rasiRNA biogenesis
Because of the repetitive nature of the genomic regions from which rasiRNAs
derive, it is unclear if rasiRNAs derive from dsRNA precursors, but recent
findings based on more extensive cloning and sequencing suggest that they have
a distinct mechanism of biogenesis that probably involves single-stranded
precursors (see Fig. 4B)
(Saito et al., 2006;
Vagin et al., 2006;
Brennecke et al., 2007;
Gunawardane et al., 2007).
Most of the current information on rasiRNA biogenesis is based on studies in
D. melanogaster. The maturation of rasiRNAs is independent of Dicer
(Vagin et al., 2006).
Processing of the rasiRNA 5' end is believed to be performed by the Piwi
proteins Piwi, Aubergine and Ago3. One model of rasiRNA 5' processing is
called the ping-pong model, in which antisense and sense rasiRNAs, associated
with the Piwi proteins Piwi/Aubergine and Ago3, respectively, guide rasiRNA
primary transcript cleavage and further participate in an amplification loop
to produce additional rasiRNAs that target transposons (see
Fig. 4C)
(Brennecke et al., 2007;
Gunawardane et al., 2007;
Nishida et al., 2007). It
remains unclear how the ping-pong mechanism is initiated. Recently, two
proteins have also been implicated in the biogenesis of the rasiRNA 3'
end: the Phospholipase D nuclease Zucchini and the RNase HII-related protein
Squash (Pane et al.,
2007).
21U-RNAs
21U-RNAs are a class of diverse, autonomously expressed small RNAs
described in C. elegans that are about 10-times less abundant than
are miRNAs (Ruby et al.,
2006). They are precisely 21 nt long, begin with a 5'
uridine monophosphate and are modified at the 3' terminal ribose. Their
biogenesis mechanism is currently not well defined, nor is their function.
There is no evidence that they are created from a dsRNA precursor, and they
originate mostly from two broad, non-coding, but distinct, regions of
chromosome IV. 21U-RNAs have a conserved upstream sequence element (also
conserved in Caenorhabditis briggsae), which could either be a
promoter or a processing signal.
scanRNAs
scanRNAs (scnRNAs) have been identified in Tetrahymena thermophila
and other protozoa. They are longer than miRNAs, 26 to 30 nt in size, and
their biogenesis is Dicer dependent. They participate in chromatin
modification, similarly to hcRNAs, leading to DNA elimination (the most
extreme form of gene silencing) during differentiation processes following
conjugation (Taverna et al.,
2002; Liu, Y. et al.,
2004; Mochizuki and Gorovsky,
2004). They are associated with the Piwi protein Twi1. Recently, a
second, smaller-sized RNA population has been described in T.
thermophila, indicating the existence of a second endogenous small RNA
pathway in protozoans (Lee and Collins,
2006).
Molecules involved in the biogenesis of distinct classes of small RNAs
Numerous biochemical studies have been conducted to understand small RNA
biogenesis. Each class of small RNAs has distinguishing features due to
different precursor structures and different mechanisms of processing.
Proteins in addition to Ago/Piwi proteins that are involved in small RNA
biogenesis include endoribonucleases, dsRNA-binding-domain (dsRBD)-containing
proteins, RNA helicases, RdRPs, and RNA methyltransferases (reviewed by
Du and Zamore, 2005;
Kim, 2005).
RNase III endoribonucleases
The key proteins required for the biogenesis of small RNAs from dsRNA
precursors are RNase III endoribonucleases (reviewed by
Conrad and Rauhut, 2002;
Patel et al., 2006). RNase
III was first discovered in Escherichia coli
(Robertson et al., 1967),
where it modulates the expression of phage, plasmid and cellular genes by its
participation in rRNA maturation. Members of the RNase III family are present
in all species (MacRae and Doudna,
2007), and, in addition to a possible role in rRNA maturation
(Wu et al., 2000), they are
required for small RNA biogenesis. Two different RNase III subfamilies have
been identified in animals and plants, Dicer and Drosha (reviewed by
Kim, 2005). The Dicer
subfamily is characterized by having an additional N-terminal RNA helicase
domain compared with the E. coli enzyme; the Drosha subfamily has a
distinct N terminus of unknown function. In plants, the biogenesis of small
RNAs is mediated by four different Dicer RNase enzymes; members of the Drosha
subfamily are absent from plant genomes. Animals generally have one Dicer and
one Drosha, except insects, which have two Dicers and one Drosha.
The involvement of Drosha and/or Dicer in miRNA and siRNA biogenesis is
described in an earlier section and is illustrated in
Fig. 2 and
Fig. 3A, respectively.
Different species have differently sized dsRNA processing products, presumably
because of Dicer protein sequence structural variation. For example, whereas
invertebrate and vertebrate miRNAs and siRNAs are between 20 and 23 nts,
Giardia intestinalis siRNAs are approximately 25 nts long
(Macrae et al., 2006), and
T. brucei siRNAs are around 24 to 26 nts
(Djikeng et al., 2001).
Moreover, in plants, 21, 22 and 24 nt RNA species are generated by different
Dicers (reviewed by Vazquez,
2006).
In D. melanogaster, the Dicer Dcr-1 is predominantly responsible
for miRNA biogenesis, whereas Dcr-2 is required for the dsRNA processing that
produces the siRNAs that mediate RNAi (see
Fig. 2 and
Fig. 3A)
(Lee et al., 2004). The
recognition of dsRNA by Dcr-1 or Dcr-2 depends on the number of mismatches in
the dsRNA precursor; perfectly paired duplexes are preferentially recognized
by Dcr-2 (Forstemann et al.,
2007; Tomari et al.,
2007). Dcr-1-processed small RNAs are preferentially loaded onto
Ago1, whereas Dcr-2-processed siRNAs are loaded onto Ago2
(Hammond et al., 2001;
Lee et al., 2004;
Okamura et al., 2004;
Matranga et al., 2005;
Miyoshi et al., 2005;
Rand et al., 2005;
Forstemann et al., 2007;
Tomari et al., 2007).
RNA helicases and dsRNA-binding proteins
Other proteins involved in the processing of dsRNA precursors include RNA
helicases and dsRBD-containing proteins. RNA helicases have been implicated in
the assembly of some small RNA-processing intermediates into effector RNP
complexes. They may also play a role in the biogenesis of piRNAs, which
probably derive from ssRNA precursors
(Klattenhoff et al., 2007).
They include the human MOV10, RNA helicase A, and RCK/p54
(Meister et al., 2005;
Chu and Rana, 2006;
Robb and Rana, 2007), the
D. melanogaster Armitage and spindle-E
(Cook et al., 2004;
Tomari et al., 2004;
Lim and Kai, 2007), and the
plant SDE-3 (silencing defective locus 3)
(Dalmay et al., 2001).
Specific dsRBD-containing proteins associate with distinct Dicers in certain
species, including plants (reviewed by
Vazquez, 2006). For example,
in D. melanogaster, the dsRBD-containing proteins R2D2 [contains two
dsRNA-binding domains (R2) and is associated with DCR-2 (D2)] and
Loquacious/R3D1 partner with Dicer, whereas Pasha partners with Drosha. In
humans, the dsRBD-containing proteins PACT (Protein activator of PKR) and/or
TARBP2 [TAR (HIV1) RNA binding protein 2], partner with Dicer, whereas DGCR8
(DiGeorge syndrome critical region 8) partners with Drosha. These
dsRBD-containing proteins may facilitate dsRNA substrate recognition, the
loading of small RNAs onto RNP complexes and the stabilization of RNase III
enzymes. The structure of the DGCR8 dsRBD-containing protein core has recently
been solved, suggesting that the DGCR8 core recognizes pri-miRNAs in two
possible orientations (Sohn et al.,
2007).
RNA polymerases
RdRPs are involved in generating dsRNA from ssRNA templates (see examples
in Fig. 3A). These templates
are the targets of siRNAs that are generated from the trigger dsRNA
(Schwarz et al., 2002;
Pak and Fire, 2007;
Sijen et al., 2007). In
nematodes and plants, such regulatory loops are responsible for generating
diffusible small RNA-silencing signals that can propagate gene silencing and
spread viral resistance (reviewed by
Wassenegger and Krczal,
2006). Direct biochemical evidence for dsRNA polymerase activity
is sparse and has been reported only for a RdRP isolated from tomato leaves
(Schiebel et al., 1993b;
Schiebel et al., 1993a).
Another RNA polymerase, RNA polymerase IV, is specific to plant genomes and is
required for the production of most siRNAs that originate from discrete
genomic loci (Zhang et al.,
2007).
RNA methyltransferases
Another protein family involved in small RNA biogenesis is the
2'-O-methyltransferases, which, depending on the species and class of
small RNA, modify the RNA 3' end. 2'-O-methylation possibly
protects small RNAs from 3' exonucleases or modulates their affinity for
binding to the PAZ domain of different Ago/Piwi proteins
(Ma et al., 2004). In plants,
all classes of small RNAs appear to be methylated by the RNA methyltransferase
HEN1 (Ebhardt et al., 2005;
Li et al., 2005;
Yu et al., 2005;
Yang et al., 2006). In D.
melanogaster, the RNA methyltransferase Pimet/DmHen1 methylates small
RNAs bound to Ago2 or to one of the Piwi proteins
(Horwich et al., 2007;
Saito et al., 2007); miRNAs,
which predominantly associate with Ago1, are not methylated. In mammals, the
RNA methyltransferase mHEN1 is a candidate for the methyltransferase activity
that modifies piRNAs (Kirino and
Mourelatos, 2007b; Kirino and
Mourelatos, 2007a; Ohara et
al., 2007). piRNAs are also 2'-O-methylated in D.
rerio (Houwing et al.,
2007).
Conclusions
Ago/Piwi proteins constitute a large family of proteins, and their numbers and the ways in which they function to regulate gene expression via small RNAs are surprisingly diverse. Given the additional complexity of cell type-specific differences in small RNA expression, complex networks of gene regulation that involve small RNAs are beginning to emerge, networks that orchestrate important regulatory cellular processes. These networks may be further regulated by the cell type-specific expression of RNA-binding proteins and RNA-processing enzymes. The study and the characterization of these networks require the development of new experimental methods and bioinformatic approaches. We believe the most productive approaches will be the generation of antibodies that are specific to Ago/Piwi protein members, and the subsequent sequencing of small RNA cDNA libraries obtained from immunoprecipitations, as well as the analysis of associated nucleic acid targets.
The mechanisms of biogenesis and function of some of the classes of small RNAs also remain to be elucidated. For example, the RNA-processing enzymes involved in the biogenesis of mammalian piRNAs have not yet been identified. Likewise, the role of the proteins implicated in rasiRNA biogenesis awaits further biochemical study.
Understanding the processes mediated by small RNAs and the networks they regulate, as well as their mechanisms of biogenesis, will allow for the design of improved gene silencing methods mediated by small RNAs. The therapeutic development of siRNAs for targeting disease genes is already ongoing, as are studies to inhibit specific miRNAs linked to disease processes. These approaches will also benefit from our improved knowledge of how the various classes of small RNAs are generated and function.
ACKNOWLEDGMENTS
We thank D. Patel for providing the crystal structure of Aquifex aeolicus Ago (Fig. 1). The authors thank all of the members of the Tuschl laboratory for their comments and critical reading of the manuscript, in particular M. Ascano, M. Hafner, M. Landthaler and J. Pena. We would also like to thank J.-B. Ma, and C. E. Rogler for helpful discussions. We apologize to colleagues whose work was not cited due to space limitations.
Footnotes
* These authors contributed equally to this work 
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K. Miyoshi, T. N. Okada, H. Siomi, and M. C. Siomi Characterization of the miRNA-RISC loading complex and miRNA-RISC formed in the Drosophila miRNA pathway RNA, July 1, 2009; 15(7): 1282 - 1291. [Abstract] [Full Text] [PDF]
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 K. C. Pang, M. E. Dinger, T. R. Mercer, L. Malquori, S. M. Grimmond, W. Chen, and J. S. Mattick Genome-Wide Identification of Long Noncoding RNAs in CD8+ T Cells J. Immunol., June 15, 2009; 182(12): 7738 - 7748. [Abstract] [Full Text] [PDF]
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N. Fahlgren, C. M. Sullivan, K. D. Kasschau, E. J. Chapman, J. S. Cumbie, T. A. Montgomery, S. D. Gilbert, M. Dasenko, T. W.H. Backman, S. A. Givan, et al. Computational and analytical framework for small RNA profiling by high-throughput sequencing RNA, May 1, 2009; 15(5): 992 - 1002. [Abstract] [Full Text] [PDF]
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G. Alsaleh, G. Suffert, N. Semaan, T. Juncker, L. Frenzel, J.-E. Gottenberg, J. Sibilia, S. Pfeffer, and D. Wachsmann Bruton's Tyrosine Kinase Is Involved in miR-346-Related Regulation of IL-18 Release by Lipopolysaccharide-Activated Rheumatoid Fibroblast-Like Synoviocytes J. Immunol., April 15, 2009; 182(8): 5088 - 5097. [Abstract] [Full Text] [PDF]
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 J. Y. Zhu, T. Pfuhl, N. Motsch, S. Barth, J. Nicholls, F. Grasser, and G. Meister Identification of Novel Epstein-Barr Virus MicroRNA Genes from Nasopharyngeal Carcinomas J. Virol., April 1, 2009; 83(7): 3333 - 3341. [Abstract] [Full Text] [PDF]
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 D. Kumari and K. Usdin Chromatin Remodeling in the Noncoding Repeat Expansion Diseases J. Biol. Chem., March 20, 2009; 284(12): 7413 - 7417. [Abstract] [Full Text] [PDF]
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 T.-C. Chang, L. R. Zeitels, H.-W. Hwang, R. R. Chivukula, E. A. Wentzel, M. Dews, J. Jung, P. Gao, C. V. Dang, M. A. Beer, et al. Lin-28B transactivation is necessary for Myc-mediated let-7 repression and proliferation PNAS, March 3, 2009; 106(9): 3384 - 3389. [Abstract] [Full Text] [PDF]
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 S. Sim, D. E. Weinberg, G. Fuchs, K. Choi, J. Chung, and S. L. Wolin The Subcellular Distribution of an RNA Quality Control Protein, the Ro Autoantigen, Is Regulated by Noncoding Y RNA Binding Mol. Biol. Cell, March 1, 2009; 20(5): 1555 - 1564. [Abstract] [Full Text] [PDF]
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A. Wilczynska, N. Minshall, J. Armisen, E. A. Miska, and N. Standart Two Piwi proteins, Xiwi and Xili, are expressed in the Xenopus female germline RNA, February 1, 2009; 15(2): 337 - 345. [Abstract] [Full Text] [PDF]
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 R. Martin, P. Smibert, A. Yalcin, D. M. Tyler, U. Schafer, T. Tuschl, and E. C. Lai A Drosophila pasha Mutant Distinguishes the Canonical MicroRNA and Mirtron Pathways Mol. Cell. Biol., February 1, 2009; 29(3): 861 - 870. [Abstract] [Full Text] [PDF]
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 B. Kaczkowski, E. Torarinsson, K. Reiche, J. H. Havgaard, P. F. Stadler, and J. Gorodkin Structural profiles of human miRNA families from pairwise clustering Bioinformatics, February 1, 2009; 25(3): 291 - 294. [Abstract] [Full Text] [PDF]
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 M. E. Dinger, K. C. Pang, T. R. Mercer, M. L. Crowe, S. M. Grimmond, and J. S. Mattick NRED: a database of long noncoding RNA expression Nucleic Acids Res., January 1, 2009; 37(suppl_1): D122 - D126. [Abstract] [Full Text] [PDF]
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J. Carte, R. Wang, H. Li, R. M. Terns, and M. P. Terns Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes Genes & Dev., December 15, 2008; 22(24): 3489 - 3496. [Abstract] [Full Text] [PDF]
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 J. S. Eisen and J. C. Smith Controlling morpholino experiments: don't stop making antisense Development, May 15, 2008; 135(10): 1735 - 1743. [Abstract] [Full Text] [PDF]
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