Biology Reference
In-Depth Information
cell proliferation and inhibit apoptosis (
Brennecke
et al
., 2003
). miR-278
similarly exhibits joint ability to promote tissue growth and inhibit apoptosis
(
Nairz
et al
., 2006
;
Teleman
et al
., 2006
), and as mentioned, miR-14 (
Xu
et al
., 2003
) and miR-263 (
Hilgers
et al
., 2010
) are also antiapoptotic.
Finally, members of the extensive K box family of miRNAs (
Lai, 2002
;
Lai
et al
., 1998
), the largest in
Drosophila
(
mir-2
family,
mir-6
family,
mir-11
,
mir-13
family,
mir-308
), share capacity to directly repress multiple members
of the proapoptotic reaper/grim/hid/sickle family (
Brennecke
et al
., 2005
).
Therefore, it seems rather common among
Drosophila
miRNAs to be able
to inhibit apoptosis and/or to promote tissue overgrowth.
These and other examples from the literature indicate that despite
propensity of animal miRNAs to subtly repress large numbers of targets,
many
Drosophila
miRNAs elicit specific and interpretable phenotypes when
misexpressed
in vivo
. Moreover, many of these effects are not predictable
from target predictions. Therefore, directed phenotypic screening for
miRNA gain-of-function phenotypes may be a profitable strategy to gain
insight into the
in vivo
activities of miRNAs.
3.3. miRNA sponges
The improvements to HR notwithstanding, it is not a trivial effort to
generate mutant alleles in
Drosophila
. Thus, an easier route toward prelimi-
nary evidence of
in vivo
miRNA function is desirable. In mammalian
systems, the vast majority of loss-of-function studies rely upon modified
antisense oligonucleotides, termed “antagomirs” (
Krutzfeldt
et al
., 2005
).
While this method is powerful and in wide use, it is also worth considering
that the large set of antagomir-induced miRNA phenotypes in
Drosophila
(
Leaman
et al
., 2005
) were not phenocopied by subsequent null alleles. The
nature of these discrepancies remains to be understood, but the possibility of
off-target effects cannot be discounted. Importantly, one can use neither the
lack of phenotypes induced by scrambled antagomirs nor the apparent
rescue by sense small RNAs, as compelling evidence for on-target inhibi-
tion. Although such criteria are popularly used to control mammalian
studies, the former would alleviate specific off-target effects while the latter
could represent a titration effect of the antagomir away from off-target
substrates. Even if antagomirs are truly specific, they have limited options
for tissue-specific delivery. Therefore, additional methods for miRNA
sequestration are desirable.
One promising strategy is to use a decoy target transcript bearing
multiple imperfect binding sites for a given miRNA, often termed as
miRNA sponge (
Fig. 8.2
B;
Ebert
et al
., 2007
). These are proposed to act
as competitive inhibitors that distract endogenous miRNAs from regulating
bona fide targets. miRNA sponges have shown efficacy in lentiviral infec-
tions of mammalian cells (
Gentner
et al
., 2009
) and were recently shown to
