Post-transcriptional regulation of gene expression can
substantially alter the outcome of the primary information
encoded on the DNA. For example, information can be added
or deleted through alternative RNA processing (splicing,
polyadenylation, and editing). The flow of information
encoded by a particular MRNA can be regulated at the level
of transport to the cytoplasm, at the level of MRNA stability
or by directing the MRNA to a subcellular location. Finally,
translation of the MRNA into protein can be regulated
at temporal, spatial, and efficiency levels. Thus, post-
transcriptional mechanisms contribute to an increase of
the proteome and account for its complex spatial and temporal
control of expression. For example, 40-60% of human genes
are alternatively spliced in at least one exon.
Post-transcriptional regulation of gene expression is
mediated to a large portion by transacting factors, proteins
that bind RNA. ELAV (Embryonic Lethal Abnormal Visual
System) of Drosophila melanogaster is the founding
member of a large family of RNA-binding proteins containing
three RRMs (RNA Recognition Motif). ELAV is pan-neurally
expressed and is one of the first signs of neuronal differentiation.
Human homologues of ELAV were shown to affect RNA- stability
and RNA-translatability by binding to AU-rich sequences
found in the three-foot UTR of some growth factor mRNAs.
ELAV, however, has been shown to be a gene specific regulator
of alternative splicing and targets identified so far
are armadillo, neuroglian, and erect
wing.
ELAV is necessary in photoreceptor neurons and sufficient
in non-neuronal wing disc cells for the expression of
Erect Wing (EWG) protein, a transcriptional regulator.
Restriction of EWG protein expression to the nervous system
is achieved through splicing of broadly expressed primary
transcripts of ewg. The role of ELAV in mediating
nervous system specific expression of EWG is most pronounced
in regulating splicing of the last intron. To show that
ELAV regulates alternative splicing of ewg directly
we have developed an in vivo system. Using a neuronally
restricted ewg rescue construct, we show that splicing
of the last intron of ewg in photoreceptor neurons
is dependent on ELAV. In the absence of ELAV, ewg
transcripts are prematurely cleaved and polyadenylated
in the last intron. Deletion analysis of the last ewg
intron narrowed the cis- sequences necessary for ELAV-
dependent processing to about 25% of the total intron
length. To complement these in vivo studies we analyzed
binding of ELAV in nuclear head extracts by UV-crosslinking
to the remaining intron, as well as to ewg three-foot
UTR sequences. ELAV binds to AU-rich elements close to
the intronic polyadenylation site, as crosslinking can
be reduced by mutations. ELAV, however, does not bind
to three-foot UTR sequences. Next, we tested intronic
ewg RNAs in a cleavage/polyadenylation assay for
in vitro processing. Consistent with ELAV being necessary
to prevent premature polyadenylation in vivo, an ewg intronic
RNA with impaired ELAV binding sites is efficiently processed
in nuclear head extract while wild type RNA is not. To
test if ELAV inhibits three-foot end formation in intron
6 in vivo, mutations impairing ELAV binding were introduced
in the ewg rescue reporter construct. Analysis of transgenic
flies carrying these reporter constructs shows that ELAV
binding is necessary to inhibit polyadenylation in ewg
intron 6 and to promote splicing in vivo. The ewg
gene encodes an essential transcription factor with homologues
in sea urchins and vertebrates, but not in yeast or
C. elegans. Although ewg RNA is broadly expressed
and alternatively spliced, only one major EWG protein
(SC3 ORF) is made. This EWG isoform is restricted to the
nervous system and transiently occurring in indirect flight
muscles. Expressing the SC3 ORF in the nervous system
is sufficient to rescue viability. Null mutant embryos
for ewg develop with no gross morphological defects,
however, fail to hatch. Electroretinograms from whole
eye clones of an ewg null allel reveal a functional
impairment in photoreceptor neurons, rescueable with the
SC3 ORF.
To define the role of EWG in neuronal function, we aimed
in identifying direct target genes of EWG, since knowing
these genes will help understanding the importance of
its unusual post- transcriptional regulation. Therefore,
RNA profiling was done using CDNA microarrays, revealing
two sets of putative target genes: metabolic genes and
genes implicated in forms of synaptic plasticity.
To analyze the role of EWG in synaptic growth and function
at the third instar neuromuscular junction, transgenes
were developed allowing the analysis of the lethal null
aliel in mosaic animals. These transgenes contain a rescue
construct flanked by FRT sites. Flipase mediated recombination
causes the loss of the coding sequence and the promotor
drives a membrane-targeted GFP- marker. Although ewg
is vital, recombinationally induced loss of the rescuing
transgene in most neurons still allows for larval and
pupal development. Emerging adult flies, however, are
severely impaired in any coordinated movement underlining
the importance of EWG for proper neuronal function.
