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Poster Full Abstracts - RNA Biology
Poster board number is above title. The first author is the presenter
351
Nonsense mediated mRNA decay (NMD) is a cellular quality control pathway that degrades mRNA which contain premature termination codons (PTCs).
The predominant role of NMD is to identify PTC containing transcripts during and target them for degradation. In addition to its quality control function,
NMD targets many native transcripts as a mechanism of post-transcriptional regulation. NMD is required during development for larval viability, possibly
due to the misregulation of native NMD targets. Very few genes have been identified as being directly regulated by NMD, and their misregulation fails to
explain the requirement for NMD during development. Identifying genes whose regulation by NMD are necessary for proper development, and the function
of these genes, may provide insight into important developmental pathways and the role of NMD in these pathways.
To identify genes regulated by NMD we performed a suppressor screen using heterozygous deficiencies. In theory, a deficiency should lower the
transcription of mRNAs within the deficiency region by half. Thus, if lethality in an NMD mutant is due to over-expression of a target gene, transcriptional
reduction by the deficiency may suppress lethality in the mutant. To perform this screen, we used a strong hypomorphic allele of a core NMD gene,
Upf2
25G
,
and were able to identify several deficiencies that suppress lethality in this mutant. Assuming that any key target will be over-expressed in NMD mutants, we
examined expression levels of genes uncovered by rescuing deficiencies using whole genome RNA expression profiling of
Upf2
25G
. Using this procedure we
have identified a candidate gene,
Arc1
, which may be a key target of NMD.
Arc1
functions in stress response pathway, and NMD may inhibit
Arc1
expression to prevent an excessive stress response, which may cause lethality. We
are currently investigating how NMD regulates
Arc1
expression, through promoter elements or transcript features, in addition to the physiological role for
Arc1
regulation by NMD in the stress response pathway.
822C
Exploring
Drosophila
Genes Involved in the Oxidative Stress Pathway and the Response to Hypergravity.
Husein Badani, Oana Marcu, Ravikumar
Hosamani, Sharmila Bhattacharya. NASA Ames Research Center, Moffett Field, CA.
In order to investigate the effects of hypergravity on the mushroom body in
Drosophila melanogaster
, it is important to monitor behavioral changes in
knockout fruit flies. The brain is known to respond to hypergravity and studies suggest that the neurons in the brain undergo oxidative stress. Genes involved
in the oxidative stress pathway are knocked out in neuronal populations of adult flies using the GAL4-UAS system to drive the RNAi for each particular
gene. Here, we exposed
Drosophila
to a hypergravity environment by centrifuging the organisms at 3g, and we monitored changes with climbing assays.
Results depicted that select experimental samples are affected by hypergravity conditions. Genetic lines that show significant alterations will be further
studied using climbing assays and behavior analysis to reveal a complete understanding of the oxidative stress pathway.
823A
Characterization of TSE and its Role in the piRNA Pathway.
Arlise P. Andress, Yanxia Bei, Richard Carthew. Molecular Biosciences, Northwestern
University, Evanston, IL.
Previous studies have shown that the nuage ensures the repression of transposable elements in female germ cells. The RNA helicase, Spindle-E (SPN-E),
localizes to the nuage and affects localization of other nuage components, such as the Piwi protein, Aubergine. It is also essential for the production or
stability of piRNAs that silence transposons. In order to gain a better understanding on how SPN-E does this, a combination of immunoprecipitation and
mass spectrometry were utilized to identify proteins that co-purify with SPN-E. One of the top copurified proteins is the subject of this presentation. We
identified a mutation in the gene encoding this protein and have named it trapped SPN-E (TSE). Mutants are female sterile and show defects in gurken
protein localization and oocyte positioning. They also show elevated expression of I-element. Intriguingly, the TSE mutant affects SPN-E protein
localization in the ovary. SPN-E protein appears localized to a disorganized nuage and is depleted in other regions of germ cells. We suggest that TSE is
required for the proper localization of SPN-E and execution of transposon silencing.
824B
Using functional proteomic approach to study Spindle-E function.
Yanxia Bei
1
, Bryan Fonslow
2
, Arlise Andress
1
, John Yates
2
, Richard Cathew
Cathew
1
. 1) BMBCB, Northwestern Univ, Evanston, IL; 2) Department of Chemical Physiology, SR11 The Scripps Research Institute La Jolla, CA.
Transposable elements impose a serious threat to genomic integrity. Animals have thus evolved a small RNA based piRNA (piwi-interacting RNAs)
mechanism to combat the replication and mobilization of transposable elements. Spindle-E (Spn-E) is an RNA helicase whose function is crucial for the
germline piRNA synthesis/stability but its molecular function is still unknown. To define Spn-E function in the piRNA pathway, we expressed a functional
epitope-tagged Spn-E protein under its own promoter in spn-e mutant flies. We then performed immunoprecipitation followed by MudPIT mass-
spectrometry to identify Spn-E interacting proteins. We found that Spn-E associates with some but not all of the known piRNA components. From these
results together with the Spn-E localization data in different piRNA mutants, we propose that Spn-E localizes to the nuage, an electron-dense perinuclear
structure that is found in all animal germ cells, in a complex that includes a few piRNA components and RNAs. From this proteomic approach, we were also
able to identify two top Spn-E interactors as novel components involved in transposable elements silencing. Mutations in genes encoding either of these
proteins changed localization of Spn-E and also resulted in the expression of transposable elements protein. One of them, TSE, will be presented in a
separate poster, abstract control #: 70643. We are currently investigating how these two proteins interact with Spn-E to suppress transposable elements in
Drosophila germ cells.
825C
Germline Silencing of Transposable Elements.
Sidney Wang, Kiri Ulmschneider, Sarah Elgin. Biology, Washington University in St Louis, St Louis, MO.
Transposon control is a critical process during reproduction. The PIWI family proteins can use a piRNA-mediated slicing mechanism to suppress
transposon activity. In
Drosophila melanogaster
, Piwi is predominantly localized in the nucleus, and has been implicated in heterochromatin formation.
Here we use female germline-specific depletion to study Piwi function. Depletion of Piwi leads to infertility and to axis specification defects in the
developing egg chambers; correspondingly, widespread loss of transposon silencing is observed. Transposon expression analysis shows that certain
transposons require Piwi for silencing but not Aubergine. Piwi-Aub double knockdown results suggest that depending on the target transposons, germline
Piwi can function either in the same pathway as Aub or independent of Aub. Germline Piwi does not appear to be required for piRNA production. Instead,
Piwi requires Aub for proper nuclear localization. We observe a loss of HP1a and H3K9me2 in germline Piwi-depleted ovaries at the promoter region of