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Poster Full Abstracts - RNA Biology
Poster board number is above title. The first author is the presenter
350
of the oocyte and establishing the body axes. Many maternal effect genes have been identified and characterized, one of these genes being
lark
. This gene
encodes a nuclear RNA-binding protein that has been shown to be essential for oogenesis. Elimination of the
lark+
maternal component results in visible
defects in the organization of the actin cytoskeleton resulting in a ‘dumping’ defect and female sterility. Since Lark is a nuclear RNA-binding protein it
likely functions by regulating RNA splicing or nuclear-cytoplasmic transport. To date, we have currently identified 38 potential RNA targets using a RIP-
CHIP approach.
Dmoesin
(
Dmoe
), an actin-binding protein, and
Transportin
(
Trn
), a proposed protein transmembrane transporter, were found to be two
potential RNA targets of Lark. We have been particularly interested in
Dmoe
as an in vivo target since it is an actin-binding protein and its protein
localization to the developing oocyte is perturbed in
lark
mutants. We have taken an RT-PCR approach to identify potential defects in RNA splicing of
Dmoe
and
Trn
in
lark
1
mutants. Here we show that expression of
Trn
and
Dmoe
during oogenesis is perturbed in the absence of
lark
maternal expression.
818B
An analysis of maternally contributed mRNAs in early Drosophila embryogenesis and germ cell specification.
Michelle A Kowanda
1
, Stephanie Yee
1
,
Eric Lécuyer
2
, Paul Lasko
1
. 1) McGill University, Montreal, Canada; 2) Institut de recherches cliniques de Montréal, Montreal, Canada.
Germline specification by the localization of particular mRNAs at the posterior of the embryo ensures the generation of primordial germ cells, also known
as pole cells, that later combine with somatic cells to create the gonads. In two screens a number of mRNAs have been found to localize at the posterior of
the embryo, some of whose function in pole cell development have not been determined. Additionally, a mechanism of active transport has been found to
localize a subset of germ plasm mRNAs to the pole cells. Active transport along astral microtubules creates RNA islands in stage 3 embryos, ensuring that
specific mRNAs are incorporated into the primordial germ cells. Interestingly, many well-characterized mRNAs that perform crucial roles in pole cell
development are recruited to RNA islands. Stephanie Yee and I have been investigating the localization of 43 D. melanogaster posterior mRNAs in two
other Drosophila species, to utilize the conservation of localization as a potential indicator of their requirement in germline development. We found so far
that at least 37 of these 43 mRNAs localized at the posterior pole in D. simulans and 25 are localized in D. virilis, with complete overlap between the
mRNAs that localize in both species. I have chosen CG18446, CG5292, Bsg25D and gcl for further studies from the list of conserved RNA island localizing
mRNAs. Preliminary studies were started on the CG18446 mutant and no pole cell defects have been observed. This research will identify novel Drosophila
primordial germ cell specification genes by investigating poorly characterized posterior mRNAs in early embryogenesis.
819C
Genome wide analysis of mRNA sub-cellular localization in embryos and larvae, with a focus on all the
Drosophila
Nuclear Receptors.
Ronit
Wilk
1,2,3
, Jack Hu
1,2
, Henry Krause
1,2,3
. 1) Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada; 2) Banting
and Best Department of Medical Research, University of Toronto, Toronto, ON, Canada; 3) Molecular and Medical Genetics, University of Toronto,
Toronto, ON, Canada.
Transcript localization controls essential biological processes, including, cell migration, cell polarity, tissue morphogenesis and neuronal function. Thus,
the sub-cellular visualization of transcripts
in situ
(in their original place) is an important tool to infer and understand their trafficking, stability, translation
and biological functions. Here, we describe the continuation of a genome-wide screen to analyze mRNA localization and expression throughout the
development of
Drosophila
embryos (
Lecuyer et al, Cell 131, 2007
) and the beginning of a similar screen in larval tissues. We use a high-resolution
fluorescent
in situ
hybridization technique that allows us to determine sub-cellular information of each transcript. This is followed by data annotation and
entry into a searchable public database
http://fly-fish.ccbr.utoronto.ca/
. To date, we have screened about 45% of the fly genome in embryonic stages: ~70%
of these transcripts are localized sub-cellularly. We have found dozens of novel RNA localization patterns and we see a high correlation between mRNA
localization and protein function. As a pilot study to initiate a genome wide screen in larval tissues, we are analyzing all
Drosophila
Nuclear Receptors.
Nuclear Receptors are ligand-regulated transcription factors that play important roles in gene regulation and control key metabolic steps and several
developmental pathways.
Drosophila melanogaster
has only 18 Nuclear Receptor genes and they all have vertebrate homologues. Despite their function as
transcription factors, preliminary findings are revealing a variety of interesting sub-cellular localization events. Our long term goal is to screen the entire
Drosophila
genome for mRNA localization in selected larval tissues and to complete the ongoing embryonic screen.
820A
The role of the NMD pathway in endogenous gene regulation.
Alex Chapin, Mark Metzstein. Human Gen, Univ Utah, Salt Lake City, UT.
The nonsense mediated mRNA decay (NMD) pathway functions to degrade mRNA transcripts harboring nonsense mutations. This regulation allows cell
to silence mutant alleles that would encode for truncated and possibly toxic proteins. Additionally, NMD also functions to degrade many native transcripts.
For instance, the wild-type transcripts of the genes
tra
and
Oda
have been shown to be directly targeted by NMD. Genetic disruption of core NMD
components leads to inviability and upregulation of a significant portion of the transcriptome. It is not currently known which of these upregulated genes are
direct targets of NMD and whether overexpression of NMD targets is responsible for inviability in NMD mutants. We have taken several approaches to
address these two questions. We have performed a tissue-specific rescue screen to look for tissues where NMD function is vital for whole animal viability.
These results indicate that in larval stages, NMD function in neuronal tissue is sufficient for viability, suggesting that critical targets of NMD may be of
neuronal origin. To identify such neuronal NMD targets, we performed RNA-seq on RNA isolated from L3 larvae harboring a strong hypomorphic allele of
the NMD gene
Upf2
. Our data show that a candidate NMD target,
Arc1
, is upregulated > 7 fold in NMD mutants.
Arc1
is expressed in neuronal tissue and is
a target of NMD in mammals. Corroborating this result, deficiencies that uncover
Arc1
rescue the subviability of NMD mutants. We are currently examining
whether
Arc1
lies downstream of NMD regulation to influence viability. To ask whether
Arc1
and other upregulated genes in our
Upf2
mutant are direct
targets of NMD, we performed a mRNA decay timecourse in S2 cells, ± cycloheximide, an inhibitor of translation and NMD. We are currently examining
whether the transcripts upregulated in
Upf2
mutants are stabilized by cycloheximide in cell culture. In addition, we are optimizing a transgenic system in
vivo to identify direct targets of the NMD pathway by identifying genes whose stabilities are reduced by reintroduction of
Upf2
in a
Upf2
mutant.
821B
A suppression screen for required targets of the nonsense mediated mRNA decay pathway.
Jonathan O. Nelson, Mark M. Metzstein. Department of
Human Genetics, University of Utah, Salt Lake City, UT.