Biology Reference
In-Depth Information
comparable proteomics approach performed with extracts from wild-type
and
let-7
mutant worms focused on a set of predicted let-7 targets to analyze
for changes in protein levels (
Jovanovic
et al.
,2010
). Each of these proteomic
studies also noted varying degrees of target mRNA destabilization associated
with the changes in protein levels. Recently, ribosome profiling was used to
compare changes in mRNA levels to polysome association, as an indicator of
translational efficiency (
Guo
et al.
,2010
). This study showed that changes in
translation largely reflected the changes in target mRNA levels, leading to the
conclusion that miRNA regulation is predominantly through target mRNA
destabilization at least in some cell types (
Guo
et al.
,2010
).
Since miRNAs form regulatory complexes with Argonaute and AIN-1/2
(GW182) proteins, target mRNAs can be detected in association with these
factors (
Beitzinger
et al.
, 2007; Easow
et al.
, 2007; Hendrickson
et al.
, 2008;
Karginov
et al.
, 2007; Landthaler
et al.
, 2008; Zhang
et al.
, 2007, 2009
). For
example, AIN-1/2 co-precipitates with the majority of established miRNA
targets in
C. elegans
, suggesting that many of the other isolated mRNAs are
good candidates for regulation by the miRNA pathway (
Zhang
et al.
, 2007
).
An advancement over the isolation of entire mRNA targets is the ability to
detect the sequence of the mRNA fragment directly bound by the miRNA
complex through a method called CLIP (cross-linking immunoprecipita-
tion) This technique uses UV-irradiation to covalently bond proteins to
nucleic acids, followed by immunoprecipitation of Argonaute complexes
and deep sequencing of directly associated sequences (
Zisoulis
et al.
, 2011
).
CLIP studies in mouse brain, mammalian tissue culture cells, and whole
worms have revealed miRISC (miRNA-induced silencing complex) bind-
ing sites on a genome-wide scale (
Chi
et al.
, 2009; Hafner
et al.
, 2010; Leung
et al.
, 2010; Zisoulis
et al.
, 2010
). This method narrows the miRNA binding
site to 50-100nt and provides biochemical evidence that an mRNA is
bound by miRISC. In
C. elegans,
many of the well-established let-7 target
sites, including those in
lin-41, daf-12,
and
hbl-1
, were detected by the CLIP
method (
Zisoulis
et al.
, 2010
). While the exact miRNA recognition site is
yet to be determined in most of the Argonaute bound sequences, several
general features of miRNA targeting emerged from these studies; sites are
predominantly in coding exons and 3
0
UTRs, seed pairing capacity is
enriched within Ago bound regions, and association of Ago with 3
0
UTR
sites is associated with target mRNA destabilization (
Chi
et al.
, 2009; Hafner
et al.
, 2010; Zisoulis
et al.
, 2010
).
3.3. Developmental role of let-7 and its targets
A common theme for targets of
let-7
regulation is a role in promoting cellular
division and self-renewal (
B
¨
ssing
et al.
, 2008
). This is consistent with the
phenotype of
let-7
mutants where the seam cells fail to terminally differentiate
at the appropriate time and instead continue dividing (
Fig. 1.3
;
Reinhart
et al.
,
