R E V I E W
Ribo-gnome: The Big World of Small RNAs
Phillip D. Zamore* and Benjamin Haley
Small RNA guides—microRNAs, small interfering RNAs, and repeat-associated small
interfering RNAs, 21 to 30 nucleotides in length—shape diverse cellular pathways,
from chromosome architecture to stem cell maintenance. Fifteen years after the
discovery of RNA silencing, we are only just beginning to understand the depth and
complexity of how these RNAs regulate gene expression and to consider their role in
shaping the evolutionary history of higher eukaryotes.
In 1969, Britten and Davidson proposed that
RNAs specify which genes are turned on and
which are turned off in eukaryotic cells (1).
Their elegant idea was that the base-pairing
rules of Watson and Crick could solve the
problem of eukaryotic gene regulation. With
the subsequent discovery of protein tran-
scription factors—there are perhaps 1850 in
humans—the idea that a diverse array of
RNA guides sets the expression profile of
each cell type in a plant or animal was
In fact, RNAs—specifically, tiny RNAs
known as Bsmall RNAs[—do control plant
and animal gene expression. Distinct classes
of these small RNAs—microRNAs (miRNAs),
small interfering RNAs (siRNAs), and repeat-
are distinguished by their origins, not their
functions Esee the poster in this issue (2)^.
One class alone, the miRNAs, is predicted to
regulate at least one-third of all human genes
(3). Small RNAs, 21 to 30 nucleotides (nt)
in length, provide specificity to a remark-
able range of biological pathways. Without
these RNAs, transposons jump (wreaking
havoc on the genome), stem cells are lost,
brain and muscle fail to develop, plants
succumb to viral infection, flowers take on
shapes unlikely to please a bee, cells fail to
divide for lack of functional centromeres,
and insulin secretion is dysregulated. The
production and function of small RNAs
requires a common set of proteins: double-
stranded RNA (dsRNA)–specific endonu-
cleases such as Dicer (4), dsRNA-binding
proteins, and small RNA–binding proteins
called Argonaute proteins (5, 6). Togeth-
er, the small RNAs and their associated
proteins act in distinct but related BRNA
silencing[ pathways that regulate transcrip-
tion, chromatin structure, genome integrity,
and, most commonly, mRNA stability. The
RNAs may be small, but their production,
maturation, and regulatory function require
the action of a surprisingly large number of
A Brief History of Small RNA
In 1990, two groups overexpressed a pigment
synthesis enzyme in order to produce deep
purple petunia flowers, but instead generated
predominantlywhiteflowers(Fig.1) (7, 8). This
phenomenon was dubbed ‘‘cosuppression’’
because the transgenic and endogenous genes
were coordinately repressed, and its discovery
quietly ushered in the study of RNA silencing.
By the end of the decade, RNA silencing
phenomena were discovered in a broad spec-
trum of eukaryotes, from fungi to fruit flies.
RNA interference (RNAi) is perhaps the best
known RNA silencing pathway, in part because
its discovery makes it possible to block ex-
pression of nearly any gene in a wide range of
eukaryotes, knowing only part of the gene’s
sequence (9, 10). Human clinical trials testing
RNAi-based drugs are currently under way.
Building on the unexpected finding that
both sense and antisense RNA could silence
gene expression in Caenorhabditis elegans
(11), the key breakthrough in RNA silencing
was the discovery that dsRNA is the actual
trigger of specific mRNA destruction, with the
sequence of the dsRNA determining which
mRNA is destroyed (9). Later, the dsRNA
was found to be converted into siRNAs—
fragments of the original dsRNA, 21 to 25 nt
in length, that guide protein complexes to
complementary mRNA targets, whose expres-
sion is then silenced (12–14). Thus, the actual
mechanism of RNAi is remarkably like an
early model for plant cosuppression, which
postulated that small RNAs derived from the
overexpressed gene might guide inactivation
of cosuppressed genes (15).
In contrast to siRNAs, which derive from
dsRNA hundreds or thousands of base pairs
long, miRNAs derive from long, largely un-
structured transcripts (pri-miRNA) containing
stem-loop or ‘‘hairpin’’ structures È70 nt in
length [reviewed in (16)]. The hairpins are cut
out of the pri-miRNA by the dsRNA-specific
endonuclease Drosha, acting with its dsRNA-
binding protein partner DGCR8 in humans or
Pasha in flies, to yield a pre-miRNA (Fig. 2)
(2). Each mature miRNA resides in one of the
two sides of the È30–base pair stem of the pre-
miRNA. The mature miRNA is excised from
the pre-miRNA by another dsRNA-specific
endonuclease, Dicer, again acting with a
dsRNA-binding protein partner, the tar-binding
protein (TRBP) in humans or Loquacious
(Loqs) in flies. The April 2005 release of the
miRNA Registry, an online database that coor-
dinates miRNA annotation, records 1650 dis-
tinct miRNA genes, including 227 from
humans and 21 from human viruses; 1648 of
these were discovered in the 21st century.
Whereas siRNAs are found in eukaryotes from
the base to the crown of the phylogenetic tree,
miRNAs have been discovered in plants and
animals and their viruses only.
Ambros and co-workers discovered the first
miRNA, lin-4, in 1993. They identified two
RNA transcripts—one small and one smaller—
derived from the lin-4 locus of C. elegans (17).
Earlier experiments showed that loss-of-function
mutations in lin-4 disrupted the developmental
timing of worms, much as did gain-of-function
mutations in the protein-coding gene lin-14.
Noting that lin-4 could form base pairs, albeit
imperfectly, with sites in lin-14, Ambros and
colleagues proposed that the 22-nt lin-4 regulates
the much longer lin-14 mRNA by multiple
RNA-RNA interactions between the miRNA
and the 3¶ untranslated region of its mRNA
target. This remarkable paper predicted the
contemporary miRNA pathway, suggesting that
the longer 61-nt transcript corresponds to a
precursor RNA that folds into a hairpin structure
from which the 22-nt mature lin-4 miRNA is
excised. Eight years later, the prescient observa-
tion that ‘‘lin-4 may represent a class of
developmental regulatory genes that encode
small antisense RNA products’’ (17) was amply
validated by the discovery that miRNAs com-
pose a large class of riboregulators (18–23).
The lin-4 miRNA was discovered 3 years
after the first reports of RNA silencing in plants
(7, 8) and 2 years before the first hint of RNAi
in nematodes (11). However, no formal con-
nection between miRNAs and siRNAs was
made until 2001, when Dicer, the enzyme that
converts long dsRNA into siRNAs (4, 24), was
shown to convert pre-miRNAs, such as the
longer 61-nt transcript from lin-4, into mature
miRNAs, like lin-4 itself (25–27).
The human genome may contain È1000
miRNAs, a few of which may not only be
unique to humans, but may also contribute to
making us uniquely human. Recent efforts to
define the entirety of this small RNA class have
uncovered 53 miRNAs unique to primates (28).
Because miRNAs are small, they may evolve
Department of Biochemistry and Molecular Pharma-
cology, University of Massachusetts Medical School,
Worcester, MA 01605, USA.
*To whom correspondence should be addressed.
R N AR N A
www.sciencemag.orgSCIENCEVOL 309 2 SEPTEMBER 2005
silencing machinery (79). Alternatively, Cid12
and Rdp1 may be components of a common
surveillance complex—and hence dependent on
each other for their stability. This complex
would contain components of two separate
pathways that protect cells against ‘‘aberrant
RNA’’—transcripts that are misfolded, in-
correctly spliced, or damaged such that they
encode truncated proteins. Favoring this view,
the budding yeast protein Trf4p, another po-
lymerase b nucleotidyltransferase, adds poly(A)
tails to misfolded tRNAs and to aberrant
mRNAs, targeting them for destruction by the
nuclear exosome, a complex of RNA-degrading
enzymes (87–89). Use of a poly(A) tail as a
degradation signal, rather than as a stabilizing
feature that promotes mRNA translation, may
be quite ancient, as bacteria use poly(A) tails to
target RNA for destruction.
Epigenetic marks play an important role in stem
cells, which must divide to yield a daughter cell
that differentiates and another that regenerates
the original stem cell. RNA silencing has
emerged as a vital regulatory mechanism for
maintaining normal stem cell pools. Mice
lacking Dicer die at embryonic day 7.5, devoid
of Oct-4–expressing cells (90); in mammals,
Oct-4 marks stem cell lineages. At least four
genes in the RNA silencing pathway are
required for germline stem cell function in
Drosophila melanogaster. Piwi, an Argonaute
protein, is required both to maintain female
germline stem cells and to promote their pro-
liferation (91). Dicer-1, which makes miRNAs
and perhaps other types of small RNAs, and its
dsRNA-binding protein partner, Loqs, are both
required for normal germline stem cell func-
tion. In the fly ovary, germline stem cells
lacking Dicer divide slowly, dramatically re-
ducing the number of eggs generated (92). In
contrast, in females mutant for Loqs in both
the soma and the germ line, germline stem
cells are lost, either because they die or be-
cause they differentiate into oocytes without
replenishing the stem cell pool (93). It re-
mains to be established whether these defects
reflect loss of miRNAs (which require the
coordinate action of Dicer-1 and Loqs for
their maturation), loss of silent heterochro-
matin, or both.
Flies lacking Ago2 contain fewer pole cells,
type flies (94). The case of ago2 mutants is
particularly instructive, because loss of Ago2—
like loss of the very first Argonaute protein
implicated in RNA silencing, worm RDE-1
(5)—was originally reported to cause no cel-
lular defects except loss of an RNAi response
to exogenous dsRNA (95). Closer examination
revealed that many aspects of early embryo-
genesis are defective, yet the flies somehow
compensate and survive (94). In particular,
ago2 mutants show defects in chromosome
condensation, nuclear division, spindle assem-
bly, and nuclear timing, all perhaps caused by a
loss of heterochromatin assembly normally
guided by an RNA silencing pathway. It re-
mains to be shown if Ago2 acts directly in the
assembly of heterochromatin by the RNA
silencing pathway, or if components common
to the RNAi and transcriptional silencing path-
ways become unstable in the absence of Ago2
protein. But these results underscore the guid-
ing principle of small RNA function: Small
RNAs play a very big role in nearly every
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a W. M. Keck Foundation Young Scholar in Medical
Research. Supported by NIH grants GM62862-01 and
GM65236-01 (P.D.Z.). P.D.Z. is a founder of and member
of the Scientific Advisory Board of Alnylam Pharmaceu-
ticals,abiopharmaceuticalcompany thatdevelops thera-
peutic agents based on RNAi.
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