Polyadenylation of Maternal RNA of Sea Urchin Eggs After Fertilization

Abstract

Between fertilization, or parthenogenetic activation, and the two-cell stage, the content of polyadenylic acid in sea urchin eggs doubles, and the increase occurs primarily in the ribosome-polyribosome fraction. The increase is due to polyadenylation of preexisting RNA molecules synthesized during oogenesis. The polyadenylation occurs in activated, enucleated merogons. It is argued that cytoplasmic polyadenylation may play a role in mobilization of maternal messenger RNA for translation and the polyadenylic acid does not subserve an exclusively nuclear function.Fertilization or parthenogenetic activation of sea urchin eggs entrains a profound series of changes in the egg, including dramatic increases in synthesis of nucleic acids and proteins, as well as complete reorganization of the structure and permeability of the cell surface (1). The increase in protein synthesis is not affected by enucleation (2) or inhibition of transcription (3). The increase in protein synthesis reflects the increased capacity of the eggs to synthesize protein, is accompanied by a gradual increase in the number (but not size) of polyribosomes (4), and occurs on mRNA templates synthesized during oogenesis. The mechanism(s) of sequestration or inactivity of untranslated maternal mRNA of unfertilized eggs and its mobilization into active polysomes after fertilization is unknown.I will report experiments that are based on the assumption that most mRNA molecules of metazoa contain sequences of poly(A) (5). First, I will show that during the 2-hr time span from activation to first cleavage (the time during which protein synthesis attains its maximum) the content of poly(A) doubles, and that the increase occurs primarily in the ribosome-polyribosome fraction of the cell. While this work was in progress, papers reporting similar conclusions appeared (6, 7). Second, the increase in poly(A) is primarily due to a polyadenylation after fertilization of RNA molecules transscribed during oogenesis. Third, polyadenylation occurs in enucleated activated merogons; hence, the reaction is cytoplasmic. Sea urchin gametes were obtained, and embryos were raised in Millipore-filtered sea water containing 50,4g/ml of streptomycin by conventional techniques. Parthenogenetic activation was done by a modified butyric acid method (9) followed by exposure for 20 min to sea water containing an extra 30 g/liter of NaCl, after which the eggs were returned to normal sea water.Merogons were obtained by layering 0.2-1.0 ml of a 20%suspension of eggs over a linear gradient constructed from 1.0 M sucrose and a mixture of equal parts of sea water and 1.0 M sucrose. Gradients were centrifuged in either an SW 39 or SW 25 rotor at 5000 X g (middle of tube) for 6-7 min, followed by centrifugation at 26,000 X g (middle of tube) for 6-10 min. The well-separated egg fragments were collected by dripping the gradients through a puncture made in the bottom of the tube and were immediately washed in normal sea water. Eggs and embryos were fractionated by washing twice in 1.5 ...

Translational Control in Oocyte Development

“…Consider, for example, cytoplasmic polyadenylation-induced translation. This process controls translation in Xenopus and Drosophila oocytes (reviewed above; see also Dworkin and Hershey 1981;Salles et al 1994; for early studies), in sea urchin eggs (Wilt 1973;Duncan and Humphreys 1984), in mouse oocytes (Tay and Richter 2001), and probably in every other metazoan as well. Cytoplasmic polyadenylation-induced translation is not restricted to oocytes; it occurs in the brain where it mediates neuronal synaptic plasticity, and learning and memory (Wu et al 1998;Alarcon et al 2004;Berger-Sweeney et al 2006), and in mitotic mammalian somatic cells where it controls cellular senescence (Groisman and Richter 2006;Burns and Richter 2008).…”

Determination of in vivo RNA kinetics using RATE-seq

“…Values are shown along with the model fit. deadenylated transcripts can persist in the cytoplasm or be readenylated in some species (Wilt 1973), we used rRNA depletion rather than poly(A) fractionation. The lack of poly(A) selection step also enables the analysis of both coding and noncoding transcripts.…”

MicroRNA Function and Mechanism: Insights from Zebra Fish

“…Deadenylation was not an indirect effect of translational repression, because nontranslatable targets were efficiently deadenylated in the presence, but not the absence, of miR-430 Mishima et al 2006). Many maternal mRNAs are deadenylated in the egg and polyadenylated upon fertilization (Slater et al 1972;Slater et al 1973;Wilt 1973;McGrew et al 1989;Richter 1999;de Moor et al 2005).…”