2002 East Coast Worm Meeting abstract 22
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Harvard University, Department of Molecular and Cellular Biology, 16 Divinity Ave. Cambridge, MA 02138
The regulation of mRNA translation is a prominent, yet poorly understood mechanism of gene control. To gain insight into the molecular mechanisms of translational control, we are investigating the translational regulation of maternal pal-1 mRNA, which encodes a conserved homeodomain protein involved in posterior patterning of the C. elegans embryo. We have focused on translational control in the distal gonad arms of adult hermaphrodites (where pal-1 mRNA is present, yet no PAL-1 protein is detected), and have gained insight into 3' UTR regulatory elements, a trans-acting factor, and the level of the translational block.
To identify translational control elements in pal-1 mRNA, we used a lacZ reporter RNA assay. We found that the pal-1 3' UTR is sufficient to repress the translation of a lacZ reporter RNA (lacZ::pal-1 3' UTR) in the distal gonad arms of adult hermaphrodites. Through deletion analysis of the lacZ::pal-1 3' UTR RNA, we identified two regions of approximately 180 nucleotides that repress distal germline expression. Subdivision of one of these regions identified a potential translational activation element as well as a 108-nucleotide germline repression element, the GRE, which is both necessary and sufficient for robust germline repression.
Through a candidate gene approach, we identified GLD-1, a maxi-KH domain protein, as a negative regulator of PAL-1 expression. In gld-1 null mutants, ectopic PAL-1 immunofluorescence is detected in the distal gonad arms of adult hermaphrodites. In addition, gld-1 can act through the GRE to repress distal germline expression; a gfp transgene under the control of the GRE is ectopically expressed in distal gonad arms when gld-1 activity is reduced through RNAi.
Finally, to begin addressing the molecular mechanism of repression, we performed polysome analysis to determine where pal-1 mRNA from the distal germline fractionates with respect to ribosomes. Multiple experiments suggest that pal-1 translation in the distal germline may be blocked after pal-1 mRNA is loaded on ribosomes. Since initiation is often the rate-limiting step in protein synthesis, the loading of pal-1 mRNA on ribosomes in the germline may allow for the rapid accumulation of PAL-1 protein in the 4-cell embryo when the translational block is relieved.