1. Abstract
An important mechanism of gene expression regulation is the subcellular localization of messenger
RNAs (mRNAs). Incorrect localization disrupts asymmetric cell division, long-term memory formation, as
well as the establishment of metazoan body patterning during early development. Highly coordinated
interactions with nuclear and cytoplasmic proteins are required for efficient transport of mRNAs to sub-
cellular regions. More recently, factors involved in Processing body (P-body) formation have also been
connected to this process. Biochemical and genetic-based data have revealed key factors associated with
an mRNA during its life cycle, but deciphering the spatial-temporal requirements of these dynamic and
ephemeral interactions can only be achieved by direct observation in vivo.
We have made significant advances over the last decade in detecting individual mRNP complexes
in vivo, using D. melanogaster egg chambers and the molecular beacon technology. The ability to co-
visualize and track mRNPs within subcellular space in real time has been an invaluable asset for studying
RNA processes. This experimental setup has resolved details behind key dynamic events, including
translational control of mRNAs during transport and spatiotemporal determination of protein-mRNA
association and disassociation.
Our novel central hypothesis is that formation of P bodies is governed by initial nucleation events via
key core-scaffold factors, followed by the recruitment of shell-client members that exhibit different
biophysical characteristics, This, in turn, coordinates the subcellular fate of maternal mRNAs as they are
transported in multi-mRNA species complexes. To this end, we initiated studies that will determine how P-
body assembly takes place and the role(s) played by P-bodies during transcript transport in D.
melanogaster egg chambers. The objective of this proposal is to characterize how multiple P-body
members and localized mRNA transcripts are spatially and temporally organized, thus giving a much-
needed level of understanding of the mechanistic links between interconnected and interdependent
processes of mRNA transport, storage, translational repression and localization important in all eukaryotic
life. By integrating the molecular beacon technology and single-molecule RNA FISH probes with advanced
imaging approaches, we will achieve the simultaneous visualization of multiple maternal mRNAs and P-
body proteins at high resolution for the first time. Using complementary biochemical and biophysical assays,
we will further sort out and classify P-body components to reveal their involvement in RNA-dependent
processes. These studies will enable us to explore a novel molecular mechanism underlying gene
expression that involving P-bodies, and thus, will have far-reaching implications beyond cell biology
research, including viral replication, tumor formation, aging and neurodegenerative diseases.
