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Poster Full Abstracts - RNA Biology
Poster board number is above title. The first author is the presenter
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transposons that require Aub for silencing. Germline HP1a depletion leads to a loss of silencing for the same set of transposons. Considering our results and
those of others, we infer that germline Piwi functions downstream of secondary piRNA production to promote silencing of some transposons via recruitment
of HP1a. On the other hand, germline Piwi could also function in silencing certain transposons independent of Aub; the mechanism there remains unclear. In
addition to
piwi
, many other genes have been implicated in piRNA-directed silencing. Two of these, Armi and Squ, have been shown to co-IP with Piwi, and
therefore might play a role in Piwi-dependent transposon silencing. Using germline specific knockdown, we are determining whether loss of these proteins
results in deregulation of the same transposons as loss of Piwi. We are also examining how the chromatin marks at transposon loci are affected in the
knockdowns. Understanding the role of
armi
and
squ
should clarify the mechanism of Piwi-dependent transposon silencing. Supported by NIH GM068388
to SCRE.
826A
The Control of Lipid Metabolism by mRNA Splicing in Drosophila.
Nicole M Chichearo
1
, Michelle E Warren
3
, Robert M Gingras
3
, Thomas Carr
2
,
Timothy Rudolph
2
, Justin R DiAngelo
3
, Alexis Nagengast
2
. 1) Dept Biology, Widener University, Chester, PA; 2) Dept Biochemistry, Widener University,
Chester, PA; 3) Dept Biology, Hofstra University, Hempstead, NY.
The fat body of Drosophila responds to different nutrient conditions and controls overall energy metabolism by regulating long-term storage of
triglycerides in structures called lipid droplets. Therefore, the fat body in the fly serves a function similar to the liver and adipose tissues in mammals. Recent
genome-wide RNAi screens in Drosophila tissue culture cells have identified mRNA splicing factors such as the Serine-Arginine (SR) domain containing
proteins B52 and U2AF-50 as playing a role in lipid droplet formation; their decreased expression results in the production of fewer lipid droplets. Using
conditional RNAi knock down experiments under GAL4-UAS control in the fat body of larvae, we have identified several early splicing factors that control
lipid storage in vivo on both high and low nutrient food sources. Larvae raised on both food sources demonstrate a visibly lean phenotype and developmental
delay with decreased expression of U1-70K, U2AF-50 or U2AF-38 and prp19 in the fat body and this lean phenotype corresponds to a significant decrease
in triglyceride levels as measured by quantitative colorimetric assays. Decreased triglyceride levels also are observed in adult females undergoing RNAi of
the same splicing factors in the fat body when raised on the high nutrient food. Interestingly, knock-down of the SR protein 9G8 in larval fat body is male
lethal on both high and low nutrient food sources but leads to increased triglycerides on low nutrient food and decreased on high nutrient food. To further
understand these defects in lipid storage, we are taking a candidate gene approach to identify potentially alternatively spliced genes important for lipid
metabolism. Through these experiments we hope to gain insight into the mechanisms underlying tissue-specific splicing in the fat body and how the
alternative splicing of important lipid metabolic genes leads to proper fat storage.
827B
A truncated
Drosophila dADAR
mRNA isoform which is evolutionarily conserved but not translated into protein potentially regulates full-length
isoform expression during embryogenesis.
John A Cook, Lea N Chhiba, Dana L Doctor, Jack C Vaughn. Zoology, Miami University, Oxford, OH.
Adenosine Deaminases Acting on RNA (ADARs) function to deaminate (edit) some adenosines to inosines in selected pre-mRNAs. The
ADAR
gene in
D.
melanogaster
(
dADAR
) produces two major mRNA transcript classes, full-length (FL) and truncated (T). The FL-class has been functionally well
characterized and encodes a catalytic protein in the adult brain, but an inactive protein in embryos. Virtually nothing is known about the T-class isoform,
which in
D. melanogaster
has been fully sequenced, lacks a catalytic domain, contains a complete open reading frame, a stop codon in intron 6, a poly(A)-
tail, a canonical poly(A)-signal, and is translated in an
in vitro
system. During fly development, levels of the two mRNA isoform classes are inversely
related. Here, we show
via
3’-RACE and sequencing that the T-class mRNA transcript is present in every
Drosophila
species studied, extending back to
those diverging 40 million years ago. In every species studied, the sequenced transcripts terminate in intron 6, have a stop codon, a poly(A)-tail, and a
poly(A)-signal. Utilization of Westerns employing a polyclonal anti-dADAR antibody produced using full-length protein shows that the FL-class isoform in
D. melanogaster
encodes a protein with the expected molecular weight at every developmental stage. Quantification of these results shows that the FL-class
protein isoform is most abundant between 0-4 h of embryo development, then gradually declines during embryogenesis. Unexpectedly, the T-class mRNA
isoform does not appear to encode a protein during any embryonic stage of development in
D. melanogaster
. This was a surprise, since this mRNA isoform
class is present in every species studied, and is highly abundant throughout embryogenesis, suggesting that it has a conserved function. Taken together, these
observations lead us to hypothesize that T-class
dADAR
mRNA may perform a novel regulatory function, perhaps playing a role in FL-class isoform
expression during embryogenesis.