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Full Abstracts – RNA BIOLOGY
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94
Messenger RNA nuclear retention as a novel facet of the DNA damage response.
Eric Lecuyer
1,2,3
, Mélanie Douziech
1
, Carole Iampietro
1
, Neal Cody
1
,
Xiaofeng Wang
1
, Moineau-Vallée Karine
1,2
, Henry Krause
4
. 1) Systems Biology Research Axis, IRCM, Montréal, QC, Canada; 2) Département de
Biochimie, Université de Montréal, Montréal, QC, Canada; 3) Division of Experimental Medicine, McGill University, Montréal, QC, Canada; 4) Donnelly
CCBR, University of Toronto, Toronto, ON, Canada.
The subcellular trafficking of mRNAs to specific destinations in the cell is emerging as an important and prevalent layer of gene regulation. While mRNA
transport is often coupled to localized translation, which enriches the encoded proteins in the corresponding region of the cell, regulated mRNA localization
may also serve as a mechanism to prevent translation of specific messages. In a previous global study of mRNA localization in Drosophila embryos, we
identified a group of zygotically transcribed mRNAs that accumulate in nuclei that are eliminated from the forming embryonic epithelium into the
underlying yolk by a process of nuclear fall-out. This serves as a quality control mechanism to eliminate damaged cells from the somatic precursor pool
during development. The mRNA nuclear accumulation phenotype is strongly induced in embryos treated with genotoxins and is dependent on the function
of the DNA-damage response regulator Checkpoint kinase-2 (Chk2). Nuclear retention blocks the translation of this group of mRNAs, which includes
transcripts encoding core histones and developmental regulatory proteins. In the case of histone mRNAs, our results suggest that nuclear targeting stems
from an interference with the function of specialized regulatory factors involved in histone transcript maturation and nuclear export. We conclude that
mRNA nuclear retention, via the inactivation of specific mRNA export pathways, represents a new layer of regulation within the DNA damage surveillance
system that is crucial for preserving genome integrity in eukaryotes.
95
Identification of chemical compounds that inhibit Ago2-mediated small RNA silencing in
Drosophila
.
Christophe Antoniewski
1
, Caroline Jacquier
1
,
Anne-Laure Bougé
1
, Fabrice de Chaumont
2
, Jean-Christophe Olivo-Marin
2
, Hélène Munier-Lehman
2
, Clément Carré
1
, Hélène Thomassin
1
. 1)
Developmental Biology Laboratory, CNRS UMR 7622 - University Pierre & Marie Curie, 75252 Paris CDX 05, France; 2) Institut Pasteur, 25 rue du
Docteur Roux 75724 Paris CDX 15, France.
MicroRNAs fulfill essential regulatory functions in metazoan development and homeostasis. Defects in miRNA silencing machinery or in miRNA
expression have been associated to development abnormalities, genetic diseases and cancers. In
Drosophila
, miRNAs are predominantly loaded in
Argonaute-1-containing RNA induced silencing complexes (RISCs), which they guide for translational inhibition or destabilization of complementary
messenger RNAs. In addition, the miRNA silencing pathway is partially overlapping with the RNAi pathways in this organism, as miRNAs may also
associate in part with Argonaute-2, the mediator of RNA interference. We set up a miRNA-repressed reporter system in which a single inducible promoter
directs the expression of the GFP fluorescent protein and of two artificial miRNAs perfectly matching the GFP coding sequences. We showed that strong
self-silencing of the resulting automiG reporter requires Drosha and Dicer-1 functions and involves exclusively the Argonaute-2 RISC complex loaded with
the anti-GFP miRNAs. Hence, automiG provides a powerful system that reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing. We used
the automiG reporter as a biosensor to screen a chemical library of 15’104 compounds and identified 29 small molecules that strongly inhibit miRNA
silencing, out of which 4 also inhibit RNA interference triggered by long double-stranded RNA. These molecules may be used to further dissect the
overlapping small RNA silencing pathways as well as to develop therapeutics of diseases linked to miRNA overexpression.
96
Forward genetic screens for genes affecting nonsense mediated mRNA decay reveal Smg6 is not an essential decay factor.
Kimberly A. Frizzell,
Shawn Rynearson, Mark M. Metzstein. Dept Human Gen, Univ Utah, Salt Lake City, UT.
Nonsense mediated mRNA decay (NMD) is a cellular surveillance mechanism that targets specific mRNAs for rapid degradation. Targets of NMD consist
primarily of mRNAs containing premature termination codons (PTCs), typically resulting from genomic mutation, frameshifts, and alternative splicing.
Evolutionarily conserved trans-acting factors involved in NMD were identified in suppressor screens in yeast and
C. elegans
. Of the six NMD genes
conserved in
Drosophila
, null mutations in
Upf1
and
Upf2
are lethal, while loss of
Upf3
or
Smg1
are viable. Alleles of
Smg5
and
Smg6
have yet to be
characterized.
To identify alleles of known NMD genes and also new genes involved in NMD, we have performed forward genetic screens of the X and right arm of the
3rd chromosome. To detect mutations affecting NMD, we use an NMD-sensitive fluorescent reporter whose expression increases in NMD mutants. We
combine this reporter with a mosaic approach, allowing us to both bypass developmental defects associated with loss of NMD, and to identify mutations in
the F1 generation, greatly increasing screen throughput. We have isolated 31 alleles on the X-chromosome which affect NMD. We have found these are all
alleles of known NMD genes
Upf1
,
Upf2
, and
Smg1
. On 3R we have isolated 13 lines. Six lines carry alleles of
Smg6
, the only known NMD factor on 3R,
and are the first alleles of
Smg6
in
Drosophila
.
Smg6
is an endonuclease that cleaves target mRNAs near the PTC, and we find all our mutations disrupt the
conserved PIN-endonuclease domain. We have found that null alleles of
Smg6
are viable and fertile, and that loss of
Smg6
has only a moderate affect on
NMD function, suggesting that Smg6 has an auxiliary role in NMD. This alters the current NMD model, which proposes Smg6 is the sole endonucleolytic
factor required for degradation, and suggests other degradation pathways may be involved in NMD
in vivo
. Additionally, we have found the other seven
lines we have identified on 3R complement
Smg6
, indicating we have isolated a new gene or genes involved in NMD.