Evidence for simultaneous derepression of messenger RNA and the guanine nucleotide exchange factor in fertilized sea urchin eggs.
Translational control was studied in extracts of Lytechinus pictus eggs and zygotes. We showed that neither mRNA nor initiation factors alone limit translation in these lysates; rather they are together rate limiting. Added globin mRNA was translated in egg and zygote lysates but overall protein synthesis did not increase significantly as the added RNA competed with the endogenous message. The lysates mimicked the in vivo response, since microinjection of globin mRNA into L. pictus eggs similarly competed with endogenous mRNAs. A number of translational components were used to determine if they would stimulate protein synthesis in these lysates. The addition of globin polyribosomes increased the level of protein synthesis. The majority of this increase was due to reinitiation of the globin mRNA, and under these conditions the level of endogenous protein synthesis in both egg and zygote extracts did not change. The addition of crude initiation factors alone did not appreciably alter the rate of protein synthesis in the egg lysates. However, in the presence of added mRNA, these initiation factors stimulated translation two- to fourfold. Of all the initiation factors tested, only the guanine nucleotide exchange factor (GEF, eIF-2B, RF) significantly increased protein synthesis when globin mRNA was present. The addition of an unfractionated initiation factor preparation further stimulated protein synthesis in the presence of added GEF and mRNA, suggesting that a component other than mRNA and GEF was also limiting in these egg lysates. Other initiation factors, including eIF-2, eIF-4A, eIF-4B, and eIF-4F, did not substitute for the component in the unfractionated initiation factor preparation. We propose that alkalinization of the cytoplasm and the subsequent activation of initiation factors and mRNAs contribute to the large stimulation of protein synthesis in echinoid eggs after fertilization. Furthermore, we discuss the possibility that the increase in NADPH at the expense of NAD+, which occurs within 3 min after fertilization, may lead to the activation of GEF.
D000818:Animals; D005260:Female; D005306:Fertilization; D020662:Guanine Nucleotide Exchange Factors; D007700:Kinetics; D010063:Ovum / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000096:biosynthesis* / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012617:Sea Urchins; D015053:Zygote / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D010448:Peptide Initiation Factors; D011506:Proteins; D012333:RNA, Messenger