The first miRNA, lin-4, was identified in 1993 in a genetic
screen for mutants that disrupt the timing of post-embryonic
development in Caenorhabditis elegans (Lee et al., 1993).
Cloning of the locus revealed that lin-4 produces a 22-
nucleotide non-coding RNA, rather than a protein-coding
mRNA (Lee et al., 1993). lin-4 represses the expression of lin-
14, which encodes a nuclear protein (Lee et al., 1993;
Wightman et al., 1993) whose concentration must be reduced
for worms to progress from their first larval stage to the second
(Rougvie, 2005). The negative regulation of lin-14 by lin-4
requires partial complementarity between lin-4 and sites in the
3?-untranslated region (UTR) of lin-14 mRNA (Ha et al., 1996;
Olsen and Ambros, 1999). It was not until 2000 that a second
miRNA, let-7, was discovered, again in worms (Reinhart et al.,
2000). let-7 functions in a manner similar to lin-4, repressing
the expression of the lin-41 and hbl-1 mRNAs by binding to
their 3? UTRs (Reinhart et al., 2000; Slack et al., 2000; Lin et
al., 2003; Vella et al., 2004). let-7 is conserved throughout
metazoans (Pasquinelli et al., 2000), and the discovery of let-
7 (Reinhart et al., 2000), together with the subsequent large-
scale searches for additional miRNAs, established miRNAs as
a new and large class of ribo-regulators (Lagos-Quintana et al.,
2001; Lau et al., 2001; Lee and Ambros, 2001), and fueled
speculation that tiny RNAs are a major feature of the gene
regulatory networks of animals. Now more than 1600 miRNAs
have been identified in plants, animals and viruses (Lai et al.,
2003; Lim et al., 2003a; Lim et al., 2003b). The human genome
alone may contain 800-1000 miRNAs, a large portion of which
may be specific to primates (Bentwich et al., 2005; Berezikov
et al., 2005; Xie et al., 2005).
miRNAs are transcribed by RNA polymerase II as primary
miRNAs (pri-miRNAs), which range from hundreds to
thousands of nucleotides in length (Cai et al., 2004; Lee et al.,
2004; Parizotto et al., 2004). Most miRNAs are transcribed
from regions of the genome that are distinct from previously
annotated protein-coding sequences (Fig. 1). Some miRNA-
encoding loci reside well apart from other miRNAs, suggesting
that they form their own transcription units; others are clustered
and share similar expression patterns, implying that they are
transcribed as polycistronic transcripts (Lagos-Quintana et al.,
2001; Lau et al., 2001; Lee et al., 2002; Reinhart et al., 2002).
About half of the known mammalian miRNAs are within the
introns of protein-coding genes, or within either the introns or
exons of non-coding RNAs, rather than in their own unique
transcription units (Rodriguez et al., 2004). Intronic miRNAs
usually lie in the same orientation as, and are coordinately
expressed with, the pre-mRNA in which they reside; that is,
they share a single primary transcript (Rodriguez et al., 2004;
Baskerville and Bartel, 2005). A very few miRNAs reside in
the untranslated regions of protein-coding mRNAs; it is likely
that these transcripts can make either the miRNA or the protein,
but not both, from a single molecule of mRNA (Cullen, 2004).
In animals, two processing steps yield mature miRNAs (Fig.
2A). Each step is catalyzed by a ribonuclease III (RNase III)
endonuclease together with a double-stranded RNA-binding
domain (dsRBD) protein partner. First, Drosha, a nuclear
RNase III, cleaves the flanks of pri-miRNA to liberate an ~70-
nucleotide stem loop, the precursor miRNA (pre-miRNA) (Lee
et al., 2002; Lee et al., 2003; Denli et al., 2004; Gregory et al.,
2004; Han et al., 2004; Landthaler et al., 2004). The efficient
processing of pri-miRNA by Drosha requires: a large terminal
loop (?10 nucleotides) in the hairpin; a stem region that is
about one helical turn longer than the slightly more than two
helical turns of the stem of the resulting pre-miRNA; and 5?
and 3? single-stranded RNA extensions at the base of the future
pre-miRNA (Lee et al., 2003; Zeng and Cullen, 2003; Zeng
and Cullen, 2005; Zeng et al., 2005). Accurate and efficient
pri-miRNA processing by Drosha requires a dsRBD protein,
known as Pasha in Drosophila, Pash-1 C. elegans and DGCR8
in mammals (Denli et al., 2004; Gregory et al., 2004; Han et
al., 2004; Landthaler et al., 2004). The resulting pre-miRNA
have 5? phosphate and 3? hydroxy termini, and two- or three-
nucleotide 3? single-stranded overhanging ends, all of which
Discovered in nematodes in 1993, microRNAs (miRNAs)
are non-coding RNAs that are related to small interfering
RNAs (siRNAs), the small RNAs that guide RNA
interference (RNAi). miRNAs sculpt gene expression
profiles during plant and animal development. In fact,
miRNAs may regulate as many as one-third of human
genes. miRNAs are found only in plants and animals, and
in the viruses that infect them. miRNAs function very much
like siRNAs, but these two types of small RNAs can be
distinguished by their distinct pathways for maturation
and by the logic by which they regulate gene expression.
microPrimer: the biogenesis and function of microRNA
Tingting Du and Phillip D. Zamore*
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605,
*Author for correspondence (e-mail: firstname.lastname@example.org)
Development 132, 4645-4652
Published by The Company of Biologists 2005
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