Biology Reference
In-Depth Information
In addition to the isolation of miRNA suppressors through traditional for-
ward genetic screens, RNAi has enabled high-throughput screening to
identify potential miRNA targets. Several transcription factors, including
daf-12,
emerged as
let-7
targets from an RNAi screen of candidate genes
that contain predicted binding sites (
Großhans
et al.
, 2005
). The ability to
detect single miRNA targets through genetic approaches suggests that miR-
NAs have a few key targets that are responsible for phenotypes associated with
loss of the miRNA or that the targets are part of interconnected pathways that
are sensitive to changes in expression of individual genes.
Various computational approaches have predicted dozens to hundreds of
let-7 targets in
C. elegans
(
Betel
et al.
, 2008; Enright
et al.
, 2003; Hammell
et al.
, 2008; John
et al.
, 2004; Kertesz
et al.
, 2007; Lall
et al.
, 2006; Miranda
et al.
, 2006; Ruby
et al.
, 2006, 2007b
). While most programs incorporate
seed pairing, RNA structure, free energy, and conservation of target sites,
the stringency of these parameters accounts for some of the variation among
the predictions (
Bartel, 2009
). Computational predictions are often tested
by fusing the target 3
0
UTR to a reporter gene and determining if it is
regulated in the presence of the miRNA. In many cases, disruption of seed
pairing between the miRNA and target is sufficient and necessary to block
regulation (
Brennecke
et al.
, 2005; Doench and Sharp, 2004; Kloosterman
et al.
, 2004; Lai, 2005
). However, some target sites appear more compli-
cated. For example, systematic analysis of the
lin-41
3
0
UTR revealed that
multiple elements within and around the two let-7 binding sites are
required for regulation (
Vella
et al.
, 2004a,b
).
Genome wide experimental approaches that analyze changes in gene
expression in response to specific miRNAs have proven effective for identify-
ing miRNA targets. After the recognition that established miRNA targets are
regulated at the level of mRNA stability (
Bagga
et al.
,2005;Giraldez
et al.
,
2006; Wu and Belasco, 2005
), microarray technology was embraced as a high-
throughput method to detect potential targets (
Huang
et al.
, 2007; Lim
et al.
,
2005; Schmitter
et al.
, 2006; Sood
et al.
,2006
). In the first of these studies,
introduction of miR-1 or miR-124 into HeLa cells resulted in downregula-
tion of genes that were enriched for complementary binding sites to the
appropriate miRNA (
Lim
et al.
, 2005
). Microarray analysis of gene expression
changes during early mouse development, as let-7 miRNA starts to accumu-
late, coupled with target prediction programs, indicates that several early
embryonic genes, including HMGA2 and IMP-1, are directly repressed by
this miRNA (
Boyerinas
et al.
,2008
). Some of these targets are reexpressed in
cancer cells when regulation by let-7 is lost (
Boyerinas
et al.
, 2010
).
Monitoring global changes in protein expression has also provided strong
evidence for target regulation by specific miRNAs. Two studies that
employed stable isotope labeling with amino acids in cell culture (SILAC)
in conjunction with mass spectrometry identified hundreds of targets regu-
lated by specific miRNAs (
Baek
et al.
, 2008; Selbach
et al.
, 2008
). A
