Biology Reference
In-Depth Information
Abstract
Although a great deal is known about the identity, biogenesis, and targeting
capacity of microRNAs (miRNAs) in animal cells, far less is known about their
functional requirements at the organismal level. Much remains to be under-
stood about the necessity of miRNAs for overt phenotypes, the identity of
critical miRNA targets, and the control of miRNA transcription. In this review,
we provide an overview of genetic strategies to study miRNAs in the
Drosophila
system, including loss- and gain-of-function techniques, genetic interaction
strategies, and transgenic reporters of miRNA expression and activity. As we
illustrate the usage of these techniques in intact
Drosophila
, we see certain
recurrent themes for miRNA functions, including energy homeostasis, apopto-
sis suppression, growth control, and regulation of core cell signaling pathways.
Overall, we hope that this exposition of
Drosophila
genetic techniques, well
known to the legions of fly geneticists and used to study all genes, can inform
the general miRNA community that focuses on other biochemical, molecular,
computational, and structural avenues. Clearly, it is the combination of these
myriad techniques that has accelerated miRNA research to its extraordinary
pace.
1. Introduction
MicroRNAs (miRNAs) are short regulatory RNAs that mediate
broad networks of posttranscriptional repression, with consequences for
diverse aspects of development and physiology (
Flynt and Lai, 2008
).
Correspondingly, there is growing appreciation of how human disease
and cancer is driven by aberrant or dysfunctional miRNA activity.
The majority of miRNAs are generated by a characteristic biogenesis
pathway (
Kim
et al
., 2009
). In animal cells, this consists of stepwise proces-
sing of hairpin precursor transcripts by the Drosha and Dicer RNase III
enzymes to yield a
22nucleotide (nt) small RNA duplex, of which one
strand is preferentially loaded into an Argonaute protein and guides
it to regulatory targets. In addition, several alternate pathways generate
miRNAs via Drosha-independent or Dicer-independent pathways (
Yang
and Lai, 2011
).
As with other classes of Argonaute-mediated small RNAs, that is,
siRNAs and piRNAs, the miRNA serves as sequence-specific guide that
recruits the Argonaute complex to target transcripts (
Czech and Hannon,
2010
). In animal cells, the key information for miRNA target identification
lies within the 5
0
end of the small RNA (the “seed”), since
7nt comple-
ments to positions 2-8 of the miRNA are capable of mediating substantial
repression (
Brennecke
et al
., 2005
;
Doench and Sharp, 2004
;
Lai, 2002
).
Consequently, miRNA targets can be predicted genome-wide via
