'Unmasking' of stored maternal mRNAs and the activation of protein synthesis at fertilization in sea urchins.
The isolation and in vitro assay of maternal mRNPs has led to differing conclusions as to whether maternal mRNAs in sea urchin eggs are in a repressed or 'masked' form. To circumvent the problems involved with in vitro approaches, we have used an in vivo assay to determine if the availability of mRNA and/or components of the translational machinery are limiting protein synthesis in the unfertilized egg. This assay involves the use of a protein synthesis elongation inhibitor to create a situation in the egg in which there is excess translational machinery available to bind mRNA. Eggs were fertilized and the rate of entry into polysomes of individual mRNAs was measured in inhibitor-treated and control embryos using 32P-labeled cDNA probes. The fraction of ribosomes in polysomes and the polysome size were also determined. The results from this in vivo approach provide strong evidence for the coactivation of both mRNAs and components of the translational machinery following fertilization. The average polysome size increases from 7.5 ribosomes per message in 15 min embryos to approximately 10.8 ribosomes in 2 h embryos. This result gives additional support to the idea that translational machinery, as well as mRNA, is activated following fertilization. We also found that individual mRNAs are recruited into polysomes with different kinetics, and that the fraction of an mRNA in polysomes in the unfertilized egg correlates with the rate at which that mRNA is recruited into polysomes following fertilization.
Kelso-Winemiller
L C
LC
Department of Zoology, University of Texas, Austin 78712-1064.
Winkler
M M
MM
D000818:Animals; D005306:Fertilization / Q000235:genetics*; D007700:Kinetics; D015336:Molecular Probe Techniques; D011132:Polyribosomes / Q000502:physiology; D014176:Protein Biosynthesis / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics*; D012270:Ribosomes / Q000502:physiology; D012617:Sea Urchins / Q000502:physiology*
D012333:RNA, Messenger