Basic HTML Version
Poster Full Abstracts - Regulation of Gene Expression
Poster board number is above title. The first author is the presenter
342
787A
Identification of regulatory elements mediating trans-interactions at metabolic loci in Drosophila melanogaster.
Xinyang Bing, Thomas Merritt.
Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada.
Trans-interactions mediate gene expression in somatic cells through interactions between homologous chromosomes. Trans-interactions have been recently
documented in Drosophila melanogaster at the Malic enzyme (Men) locus, and we have evidence that similar interactions modify gene expression at the
Triose phosphate isomerise (Tpi) locus. To identify regulatory elements responsible for trans-interactions, we continue to annotate the regulatory regions of
genes that do, and do not, show these trans-interactions. We use P-element excision mediated mutagenesis to create deletion mutant alleles varying in size
and location, and quantify the effect of these deletions on gene expression, protein level and activity when the mutant alleles are in trans to wild-type alleles.
Putative regulatory elements are predicted in silico, and mapped to deletions in mutant alleles. Significant deviations from expected gene expression (i.e.
50% wildtype in heterzygotes) of each mutant allele can then be attributed to these deleted elements. Identification of regulatory elements mediating trans-
interactions will be systematically completed for both Men and Tpi loci. We are concurrently annotating the regulatory regions of two genes that do not
appear to exhibit trans-interactions (isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase). Comparison of regulatory elements across these four
loci will identify elements that are, and are not, involved in trans-interactions, allowing us to draw conclusions about the regulatory elements that are critical
to trans-interactions, and improving our understanding of the specific mechanisms by which these trans-interactions are mediated or precluded in D.
melanogaster.
788B
REMSA and mutational analysis reveal a novel role for full-length dADAR in
Drosophila rnp-4f
5’-UTR alternative splicing regulation during
embryogenesis.
Sushmita Ghosh, Girija Lakshmi, John Cook, Gabriel Jones, Roshni Parikh, Bridgette Rawlins, Jack Vaughn. Zoology, Miami University,
Oxford, OH.
The
Drosophila rnp-4f
gene encodes a splicing assembly factor that dimerizes U4- and U6-snRNPs during spliceosome formation. 5’-UTR pre-mRNA
intron processing results in two major isoforms, unspliced and alternatively spliced. The unspliced isoform has a secondary structure where an intron pairs
with adjacent highly evolutionarily-conserved exon 2 to form a stem-loop. The coding potential for the two isoforms is identical, raising interesting
questions as to the control mechanism and functional significance of this 5’-UTR intron splicing decision. It is known that the unspliced isoform localizes
largely in the developing fly central nervous system, as do
dADAR
mRNAs, and that dADAR uses dsRNAs for substrate during A-to-I RNA editing. These
observations suggested an hypothesis in which dADAR protein may bind to the
rnp-4f
pre-mRNA stem-loop and inhibit splicing. To test this hypothesis,
RNA Electrophoretic Mobility Shift Assay (REMSA) was carried out using
in vitro
transcribed stem-loop RNA incubated with embryo protein extract. Two
RNA-protein complexes are detected by shifted RNA bands. Protein extract from a
dADAR
null mutant fly line results in only one shifted band, and
recombinant dADAR results in a band shift. A mutated stem-loop in which the conserved exon 2 sequence is changed but secondary structure maintained
results in diminished band shifts. To determine if unspliced mRNA levels are correlated with presence of dADAR protein during embryo development, qRT-
PCR was carried out using embryo protein extracts from wild-type and the
dADAR
mutant. A dramatic decrease in unspliced mRNA levels occurs in the
dADAR
mutant, a finding consistent with the REMSA results. These observations demonstrate a novel non-catalytic role for dADAR protein in
rnp-4f
5’-
UTR alternative intron splicing regulation. A model is proposed to explain the results. We are now attempting to identify the regulatory protein(s) which
bind to the stem-loop using MALDI-TOF technology.
789C
The role of
Drosophila
ATF4(crc) in the Unfolded Protein Response.
Min-Ji Kang, Josepher Li, Dowhan Kim, Hyung Don Ryoo. Dept Cell Biol, New
York Univ Sch Med, New York, NY.
Stress in the endoplasmic reticulum (ER) activates transcriptional response pathways, widely referred to as the Unfolded Protein Response (UPR). These
pathways require transmembrane proteins that can detect stress in the ER, and transmit that information to the cytoplasm. In mammals, three pathways are
particularly well-established, mediated by ER-stress sensors, ATF6, IRE1 and PERK. Upon detection of ER-stress, PERK phosphorylates eIF2alpha, which
leads to a selective activation of ATF4 translation to induce the Unfolded Protein Response. Here, we report the function of
Drosophila
ATF4. This protein
is encoded in the cryptocephal (crc) locus, and is essential for development.
Drosophila
ATF4 has two upstream open reading frames (uORFs) that are
inhibitory and normally block downstream ATF4 expression. Upon ER-stress, or other conditions that lead to eIF2alpha phosphorylation, ribosomes can
bypass such uORFs to synthesize ATF4. Using this feature, we developed the ATF4 reporter, which is activated by misexpression of misfolded Rhodopsin-1
that is a model for Autosomal dominant retinitis pigmentosa (ADRP). Once activated,
Drosophila
ATF4 stimulates the transcriptional activation of XBP1, a
mediator of the IRE1 branch of the UPR. In addition, ATF4 induces Thor that suppresses cap-dependent protein translation. By enhancing UPR and
reducing cap-dependent translation, ATF4 is thought to relieve unfolded protein overload in the ER, not only in response to excessive stress, but also during
normal development.
790A
Sub-type specific regulation of
Drosophila
glutamate receptor production by the novel receptor mRNA associated genes
optimus-prime
(
opr
) and
bumblebee
(
bbe
).
Julie E. Karr
1
, Subhashree Ganesan
2
, Magdalena M. Paces
3
, David E. Featherstone
4
. 1) Science and Mathematics , Columbia College
Chicago, Chicago, IL; 2) Neurosciences Institute, Stanford School of Medicine, Stanford, CA; 3) Loyola University Chicago, Chicago, IL; 4) Biological
Sciences, University of Illinois at Chicago, Chicago, IL.
Postsynaptic receptor abundance is a critical determinant of synapse strength. We are identifying and studying mechanisms that control glutamate receptor
(GluR) abundance in
Drosophila
embryonic/larval neuromuscular junctions (NMJ). One process that appears particularly important is regulation of the
production, trafficking, stability, and translation ofGluR mRNA. GluR subunit mRNA in embryonic/larval NMJs is associated with messenger
ribonucleoprotein (mRNP) complexes distributed throughout the cytoplasm of postsynaptic muscle cells. Here, we show two novel proteins that appear to
associate specifically with
GluRIIA
mRNA and regulate GluRIIA protein abundance. The first of these genes is CG12149, which we named '
optimus-prime
(
opr
)'. Mutants and muscle-specific RNAi knockdown of
opr
leads to loss of GluRIIA protein but no change in
GluRIIA
mRNA quantity or loss of other
GluR subunits. A polyclonal antibody raised against Opr shows immunoreactivity distributed throughoutmuscle cells, but Opr is not seen at motor neuron