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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
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