| Most
of the work, to date, on the eye specification cascade has
been centered primarily upon the question of how each gene
within the network is regulated at the level of transcription.
These efforts have led to a greater appreciation of the regulatory
complexity that exists within the retinal determination network.
In the last 10 years a new layer of regulation has been identified,
namely the regulation of mRNA stability and translation by
small non-coding RNAs (ncRNAs). Several different classes
of regulatory ncRNAs have been identified: siRNAs, piRNAs,
rasiRNAs and miRNAs. The focus of my research is how miRNAs
(or micro RNAs) affect the earliest decisions in eye development.
A search of extant databases has identified a number of miRNAs
that are predicted to target the 3`UTRs of several eye specification
mRNAs (Figure 1).
Each miRNA is
first transcribed and folded into a 70nt hairpin precursor.
Like all miRNA precursors the ones that target the eye specification
transcripts are predicted to be (1) initially processed and
exported from the nucleus to the cytoplasm; (2) further processed
by DICER into a mature 22-23nt miRNA and; (3) then bound to
its target sequence by the RISC complex thereby initiating
translational repression (Figure 2). Each of the miRNAs of
interest are predicted to recognize and base pair with the
3` UTR region of its target mRNA. The RISC complex mediates
the formation of a double stranded RNA species that consists
of the miRNA bound to its target sequence
(Figure 2).
As
a first step towards characterizing the role that each miRNA
plays in retinal determination I have cloned and expressed
each miRNA within the developing eye using ey-GAL4
and GMR-GAL4 drivers. The ey enhancer directs expression
ahead of the advancing morphogenetic furrow while the GMR
element promotes expression in all developing photoreceptor
cells. If the miRNAs are in fact targeting transcripts of
the eye specification genes then forced expression of the
miRNAs are predicted to mimic phenotypes seen in loss-of-function
RD mutants. Our preliminary results suggest that expression
of many but not all miRNAs do in fact alter eye development
(Figure 3). I am currently in the process of conducting a
careful analysis of larval eye development in each forced
expression condition.
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