Biology Reference
In-Depth Information
4.3. Molecular analysis of Ecdysone- and JH-control
of let-7-C, miR-34, miR-8, and miR-14 miRNAs
A key question has been the identity of the transcriptional regulators of these
miRNAs. Ecdysone induction of
miR-100
,
let-7
, and
miR-125
suggested
that these miRNAs might be regulated either by the 20E receptor itself
or by one of its three well-established direct targets,
Ecdysone-induced protein
74EF
(
Eip74ef
),
Ecdysone-induced protein 75B
(
Eip75b
), and
broad
(
br
). The
co-regulation of these miRNAs is not surprising, since these miRNAs are
co-transcribed from a single locus termed the
let-7-Complex
(
let-7-C
)
(
Sokol, Xu, Jan, & Ambros, 2008
). Two pieces of additional data, however,
suggested a potentially more complex relationship between Ecdysone and
let-7-C
miRNAs (
Bashirullah et al., 2003
). First, expression of mature
processed
let-7-C
miRNAs is delayed relative to known direct targets,
suggesting that the
let-7-C
locus is not a direct target of the 20E receptor.
Second, silencing of
EcR
using a heat-shock inducible RNAi transgene
during late larval development did not affect
let-7-C
miRNA expression
in vivo
, even though the expression of known direct targets was severely
reduced. These data raised the possibility that the Ecdysone responsiveness
of the
let-7-C
locus was independent of 20E and the 20E receptor.
Chawla and Sokol, however, provide clear supporting evidence that the
let-7-C
locus is a direct target of the 20E receptor (
Chawla & Sokol, 2012
).
Consistent with this notion, the primary
let-7-C
transcript is detected within
30 min of 20E treatment of cultured cells. The delayed expression of mature
miR-100
,
let-7
, and
miR-125
observed in previous experiments is likely due
to their post-transcriptional processing. The 20E responsiveness of the
let-
7-C
locus is mediated by three conserved sequence motifs that matched
the known 20E responsive elements site and that are bound by recombinant
EcR and Usp heterodimers
in vitro
. In addition, these same sequences are
responsible for
let-7-C
expression
in vivo
, since rescuing transgenes in which
they were deleted displayed reduced nervous system expression and rescuing
activity. Taken together, this data indicates the 20E-receptor directly acti-
vate the
let-7-C
locus during development. The observation by Bashirullah
et al. that
let-7-C
miRNAs are activated even when
EcR
is knocked down by
RNAi may indicate that these miRNAs are responsive to low levels of EcR,
since
EcR
RNAi is unlikely to completely eliminate EcR (
Bashirullah et al.,
2003
).
let-7-C
expression and activity are not completely eliminated in
transgenes in which the 20E-receptor binding sites are deleted, however,
indicating that the
let-7-C
locus is independently regulated by additional
unknown factors that await identification.
