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Full Abstracts – RNA BIOLOGY
153
91
oskar
translational activation: a 5’ activating region directs translational activation throughout the oocyte.
Matt Kanke, Goheun Kim, Young Hee
Ryu, Paul Macdonald. Molecular Cell and Developmental Biology, University of Texas at Austin, Austin, TX.
Embryonic patterning is dependent on proper regulation of
osk
RNA expression.
osk
is transcribed in the nurse cells, transported to the oocyte, and
subsequently localized to the posterior of the oocyte. A program of translational repression and activation restricts Osk protein accumulation to the posterior
of the oocyte. Repression is mediated by Bruno, which binds to sites in the
osk
mRNA 3' UTR. Activation relies on multiple types of regulatory elements,
positioned in a 5' region and in the 3' UTR. The element(s) in the 5' region, which is defined by large deletion and inversion mutations, is thought to act only
at the posterior of the oocyte (Gunkel et al., 1998, Localization-dependent translation requires a functional interaction between the 5’ and 3’ ends of
oskar
mRNA.
Genes Dev.
12, 1652-1664). To better understand the role of the 5' region we tested a series of GFP reporter transgenes, with or without the
osk
5'
region. Surprisingly, inclusion of the
osk
5' region causes enrichment of GFP throughout the oocyte, not just at the posterior pole. Oocyte enrichment is
abolished by the 62 nt inversion mutation that defines the 5' regulatory element. The inversion mutation has a much lesser effect on GFP levels in the nurse
cells. Thus, the
osk
5' region provides one or more elements that specifically activate translation in, and throughout, the oocyte. We suggest that expression
of
osk
specifically at the posterior relies on at least two different forms of activation. One makes the mRNA translationally competent throughout the oocyte;
this form of activation is obscured in the
osk
mRNA by concomitant Bruno-mediated repression. A second form of activation involves the release from
Bruno repression specifically at the posterior of the oocyte, allowing for accumulation of Osk protein.
92
Local trafficking sorts germ plasm RNPs into distinct cortical domains.
Jack J. Lee, Kristina S. Sinsimer, Shawn C. Little, Stephan Y. Thiberge, Eric F.
Wieschaus, Elizabeth R. Gavis. Princeton University, Princeton, NJ.
RNA localization is a conserved mechanism for generating cell and developmental polarity. Although different mRNAs may have overlapping patterns of
localization, it is not known whether transcripts with similar destinations are co-transported. In the process of germ plasm assembly that occurs during late
stages of
Drosophila
oogenesis,
oskar (osk)
and
nanos (nos)
mRNAs accumulate concurrently at the posterior oocyte cortex using a common localization
machinery, suggesting that they might co-habit the same transport particles. Using a combination of live imaging and high sensitivity fluorescence in situ
hybridization to visualize
osk
and
nos
at high resolution, we find that the two mRNAs form distinct RNP particles within the oocyte cytoplasm. Upon
localization at the posterior cortex,
nos
, but not
osk
, associates with Vasa (Vas) protein and the two types of RNPs occupy non-overlapping domains. To
explore the basis for these unique distributions, we visualized fluorescently labeled
osk
,
nos
, and Vas in live oocytes using 4D two-photon microscopy.
While previous studies have shown that posterior localization of germ plasm RNPs is maintained by the actin cytoskeleton, this high-resolution analysis
revealed surprisingly that localized
osk
and
nos
RNPs undergo directed movements. Mutational and pharmacological analyses showed that this motility is
microtubule and dynein-dependent. When dynein function is disrupted,
osk
and
nos
RNPs overlap, indicating that the observed motility is necessary for the
segregation of
osk
and
nos
RNPs. This dynein-dependent organization of
osk
and
nos
RNPs in distinct domains is maintained into embryogenesis. During
embryogenesis,
nos
is incorporated into germ cell progenitors, where it functions in germline development. In contrast,
osk
is not enriched in the germ cells,
consistent with its prior role in nucleating germ plasm in the oocyte. We propose that local trafficking at the posterior cortex of the late oocyte sorts germ
plasm RNPs into distinct domains that facilitate their functions in germline development.
93
The Drosophila pan gu kinase complex regulates RNP stability via ubiquitin-dependent proteolysis.
Risa Broyer
1
, Brian Sato
2
, Jim Wilhelm
1
. 1)
Section on Cell and Developmental Biology, UC San Diego, La Jolla, CA; 2) Dept of Molecular Biology and Biochemistry, UC Irvine, Irvine, CA.
While mRNA is considered to be quite labile, maternal mRNA is highly stable with a half-life greater than two weeks in the oocytes of some organisms.
This stability is thought to be due to the protective effects of the protein subunits of maternal RNA-protein (RNP) complexes. While an asset when the
oocyte is stockpiling transcripts to drive early embryogenesis, this mRNA stability poses a problem for the maternal to zygotic transition when many
maternal messages are degraded to shift to zygotic transcriptional control. Previous studies have focused on defining the pathway for maternal mRNA
degradation: activation of the protein kinase, Pan gu, triggers translation of the RNA binding protein, Smaug, which in turn recruits the CCR4/Twin
deadenylase causing message destruction. However, these studies also demonstrated that there is an additional pan gu dependent branch in the pathway that
is required for mRNA degradation. We have found that in addition to regulating mRNA stability, pan gu is required for ubiquitin-mediated degradation of
three subunits of the maternal RNP complex - the translational repressor, Cup, the RNA helicase, Me31B, and the LSm domain protein, Tral. In order to
identify components of this pan gu dependent, smaug-independent pathway, we screened for mRNA degradation mutants where Smaug protein was
expressed normally. This screen identified numerous regulators and components of the meiotic anaphase promoting complex, an E3 ubiquitin ligase. All of
these mutants were defective in ubiquitin-mediated degradation of RNP subunits consistent with the meiotic APC being an important regulator of RNP
stability. Furthermore, this effect is unlikely to be secondary to cell cycle arrest since meiosis is completed in one of our APC regulators. These results
suggest that mRNA stability in the early embryo is regulated by both activating mRNA degradation factors, such as Smaug, and removing RNA stabilizing
factors via ubiquitin mediated proteolysis.