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conserved miRNA seed matches (
Bartel, 2009
); location within the tran-
script, local sequence bias and secondary structure, and other RNA-binding
proteins can also influence target efficacy. The abundance of sequenced and
aligned genome alignments provides evidence that a large fraction of well-
studied metazoan transcripts bear conserved miRNA target sites (
Friedman
et al
., 2009
;
Jan
et al
., 2011
;
Mangone
et al
., 2010
;
Ruby
et al
., 2007b
). The
endogenous impact of miRNA-mediated repression should be larger still,
in light of the facts that many functional sites are not conserved and that at
least certain types of nonseed sites can confer repression (
Brodersen and
Voinnet, 2009
).
The wealth of information from comparative genomics, as well as
transcriptome- and proteome-based analyses (
Baek
et al
., 2008
;
Guo
et al
.,
2010
;
Lim
et al
., 2005
), provides ever-increasing information on the scope
of miRNA-mediated repression. Nevertheless, such studies have not
provided a straightforward route toward predicting the phenotypic con-
sequences of altering miRNA activity in the context of the whole organism
(
Smibert and Lai, 2008
). Ironically, some of the best-understood biological
usages of miRNAs derived from studies conducted prior to the formal
recognition of miRNAs. In particular,
Caenorhabditis elegans
genetics per-
mitted the first (lin-4) and second (let-7) identified miRNAs to be placed
within regulatory hierarchies that control developmental timing and iden-
tified their key direct target genes (
Lee
et al
., 1993
;
Moss
et al
., 1997
;
Reinhart
et al
., 2000
;
Wightman
et al
., 1993
). In addition, genetic studies
of the
Drosophila
Notch pathway identified key miRNA target genes prior
to the cloning of miRNAs (
Lai and Posakony, 1997, 1998
;
Lai
et al
., 1998
)
and led to the concept of 7nt complements to miRNA 5
0
ends as animal
miRNA-binding sites (
Lai, 2002
). Therefore, genetic analysis was central to
revealing the existence and mechanism of miRNAs.
The genome and transcriptome of
Drosophila melanogaster
have been
extensively scoured for miRNA genes, and its current state of annotation
is perhaps the deepest among any animal species (
Berezikov
et al
., 2011
;
Chung
et al
., 2011
) and includes loci derived from several noncanonical
pathways (
Flynt
et al
., 2010
;
Okamura
et al
., 2007
;
Ruby
et al
., 2007a
).
Deletion mutations of over 30 well-conserved
Drosophila
miRNA genes,
comprising 18 genomic loci/clusters, have been described and collectively
reveal critical biological requirements for miRNAs. Many other miRNA
loci have been associated with compelling gain-of-function phenotypes,
and still others are “interesting” from the point of view of cell- or tissue-
specific expression patterns (
Aboobaker
et al
., 2005
;
Ruby
et al
., 2007b
),
conserved targeting of well-studied protein-coding genes (
Ruby
et al
.,
2007b
;
Stark
et al
., 2007
), or principles that relate cohorts of miRNA target
genes (
Lai, 2002
;
Stark
et al
., 2005
). Altogether, studies in the
Drosophila
model have richly illuminated our understanding of miRNA-mediated
regulation (
Smibert and Lai, 2010
).
