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the 5p product predominates for
miR-iab-4
and is almost exclusively
expressed for
miR-iab-8
(
Aravin
et al
., 2003
;
Bender, 2008
;
Ruby
et al
.,
2007
;
Stark
et al
., 2008
;
Tyler
et al
., 2008
).
5.2. Integrating developmental expression with
target predictions
The rostral boundaries of
Drosophila miR-iab-4
and
miR-iab-8
expression are
consistent with colinearity. Their expression patterns are nonoverlapping
with one another and are largely complementary to predicted and known
Hox targets, which include more proximal (3
0
) Hox mRNAs (
Table 2.1
)
(
Bender, 2008
;
Ronshaugen
et al
., 2005
;
Stark
et al
., 2008
;
Tyler
et al
.,
2008
). Further, it has been noted that the mutually exclusive expression of
miR-iab-4
and
miR-iab-8
could be generated or reinforced through mutual
repression, perhaps via transcriptional interference or posttranscriptionally
through annealing of transcripts to produce of double-stranded RNA (
Stark
et al
., 2008
).
miR-iab-4
is expressed in precursors of segments A2-A7 (parasegments
8-12) at the cellular blastoderm stage and becomes refined to a subset of
ectodermal cells during germband extension. Ectodermal expression over-
laps with Abd-A, but levels are inverse to the validated target Ubx in
segments where they are coexpressed, with areas of highest
miR-iab-4
corresponding to areas of lowest Ubx protein. Although
miR-iab-4
expres-
sion is highest in embryos, processed transcripts are present in larvae and
adult (
Bender, 2008
;
Ronshaugen
et al
., 2005
;
Stark
et al
., 2008
).
miR-iab-8
is expressed in the precursors of segments A8-A9 (paraseg-
ments 13-14), with similar timing to
miR-iab-4
. Expression in late embryos
becomes refined to the CNS within these segments. Although primary
transcripts can be detected by RT-PCR at all stages, processed
miR-iab-
8
appears limited to late embryos and early larvae. In contrast to
miR
-
iab-4
,
miR-iab-8
is expressed only in the posterior segments, overlapping with
Abd-B, but where its other predicted and known targets (
Antp, Ubx,
and
abd-A
) are not translated at all (
Bender, 2008
;
Lewis, 1998
;
Stark
et al
., 2008
;
Tyler
et al
., 2008
).
In
D. melanogaster
,
the Ubx
3
0
UTR bears seven predicted binding sites
for
miR-iab-4
-
5p
, five or six of which are also predicted to bind more
strongly to
miR
-
iab-8-5p
. Most sites are conserved across
Drosophilids
, and
some across arthropods (
Bender, 2008
;
Miura
et al
., 2011
;
Ronshaugen
et al
., 2005
;
Stark
et al
., 2003, 2008
;
Tyler
et al
., 2008
). Although individual
target sites evolve rapidly, site variation (at least within
Drosophila
) appears
compensatory, such that loss or weakening of one binding site often
accompanies gain or strengthening of another. Such changes might be
predicted to maintain a similar level of regulation of
Ubx
across species
(
Ronshaugen
et al
., 2005
;
Stark
et al
., 2008
).
In vivo
reporter assays have