Program of the 2001 International Worm Meeting - Sternberg Lab ...

956
956. Role of MEX-3 in Patterning
PAL-1 Expression
Michael C. Fitzgerald, Craig P.
Hunter
Department of Molecular and Cellular Biology,
Harvard University, Cambridge, MA 02138
PAL-1 is a caudal-like homeodomain protein
that is first expressed at the 4-cell stage in the
two posterior blastomeres. This spatial and
temporal patterning of PAL-1 expression is
dependent on the putative RNA binding protein
MEX-3 and the pal-1 3’ untranslated region
(UTR). A large body of circumstantial evidence
suggests that MEX-3 may repress pal-1
translation by binding to the pal-1 3’ UTR. We
are taking a biochemical approach to test this
possibility directly. Current efforts focus on
using reversible RNA-protein cross-linking,
followed by immunoprecipitation of MEX-3,
and RT-PCR analysis of the co-precipitated
RNA. Positive results in this assay will be
followed by experiments to investigate the
mechanisms by which MEX-3 patterns PAL-1
expression.
957. Identification of factors involved
the translational control of GLP-1 in
the Caenorhabditis elegans early
embryo
Veronica Marin 1 , Lauren Aveline 2 ,
Kathyrn Resing 2 , Natalie Ahn 2 ,
Thomas C. Evans 1
957
1 Department of Cellular and Structural Biology,
University of Colorado Health Sciences
Center, Denver, CO
2 Department of Biochemistry, University of
Colorado, Boulder, CO
GLP-1 is a Notch family receptor that is
important in cell fate specification of the early
Caenorhabditis elegans embryo. The translation
of glp-1 is spatially and temporally regulated
through sequences in its 3 untranslated region
(UTR). GLP-1 protein is translated mostly in
anterior cells beginning at the two-cell stage
continuing to the 16-cell stage. Several
regulatory regions of glp-1 3 UTR have been
identified. There is one region at the 3 end of
the UTR that is essential for translational
repression in oocytes and 1-cell embryos. This
region is called the oocyte control region
(OCR). Another region is essential for the
special regulation of GLP-1 translation. This
GLP-1 localization region (GLR) is both
necessary and sufficient for the spatial
regulation of GLP-1. The GLR contains both an
element necessary for translational repression in
posterior cells and another element required for
activation of translation in the anterior cells.
Our hypothesis is that RNA binding proteins
interact with the 3’ UTR of glp-1 controlling its
translation, and that these proteins contribute to
anterior-posterior patterning. In order to identify
these proteins, we have performed RNA
precipitations to identify proteins that bind the
regulatory regions of the glp-1 3 UTR. These
RNA precipitations have isolated two bands
which can be visualized by UV crosslinking,
p58 and p30, that bind with specificity to a
region within the GLR that is required for
repression of translation in posterior cells. These
two bands are not able to crosslink to a probe
containing a 5-nucleotide mutation within this
repression region. Furthermore, these bands
cannot be competed off with an excess of this
same mutation. We conclude that p58 and p30
are RNA binding proteins that may be involved