Stability and movement of mRNAs and their encoded proteins in Xenopus oocytes.

Abstract

The stability and movement of several polyadenylated (poly A ÷) and nonpolyadenylated (poly A-) mRNAs in Xenopus oocytes have been examined. At least 50% of the poly A ÷ mRNA molecules (9S rabbit globin mRNA, chicken ovalbumin, and lysozyme) were stable in oocytes over a 48-h period, irrespective of the amount injected. About 50% of injected poly A-reovirus mRNAs was degraded within the first 24 h of injection, irrespective of the amount injected, although no further degradation was observed over an additional 24 h.The movement of all poly A + mRNAs injected at either the animal or vegetal pole of the oocyte was very slow. Little movement of RNA from the animal half to the vegetal half was observed even 48 h after injection. In contrast, similar amounts of mRNA were present in both halves 48 h after vegetal pole injection. Similar results were obtained after injection of poly A-reovirus mRNAs.The movement of the proteins encoded by the poly A ÷ mRNAs was studied in the 6-h period after injection when little mRNA movement had occurred. 85% of the globin synthesized accumulated in the animal half irrespective of injection site. The movement of the sequestered secretory proteins ovalbumin and lysozyme in the same oocytes as globin was much slower; very little lysozyme appeared in the half of the oocyte opposite the site of injection.The large size and ample store of molecular components have made the amphibian oocyte a popular choice for the assay of biological materials after microinjection. The oocyte has proved an excellent vehicle for investigation of mRNA translation (1-3), DNA transcription (4, 5), RNA splicing (6), chromatin assembly (7, 8), protein segregation and secretion (9, 10), and nucleocytoplasmic movements of RNA (11). When you consider the large size of the oocyte (>1.2 mm), it is surprising that there has been no systematic study of the rate of movement of injected macromolecules (except polysaccharides [ 12]) from the site of injection, although this could be because considerations of macromolecular diffusion rates are of limited significance for many of the uses cited above. However, in several studies the extent of competition of injected mRNAs with endogenous mRNAs has been used to investigate spare translational capacity (13) or relative mRNA translational efficiencies (14, 15), or to assess the translational status of maternal mRNA pools (16). Richter and Smith (16) have also used co-injection or sequential injections of different RNAs to demonstrate the existence of distinct translational pools in oocytes for secretory and nonsecretory mRNAs. In the quantitative interpretation of all the competition data cited above, it is implicitly assumed that injected mRNAs diffuse rapidly within the oocyte to achieve a homogenous equilibrium distribution. However, various studies attest to the complex spatial arrangement of the endogenous macromolecules in the oocyte. For example, several groups demonstrated distinct gradients of specific classes of RNA within oocyte and fertilized egg cytopla...

Improperly Terminated, Unpolyadenylated mRNA of Sense Transgenes Is Targeted by RDR6-Mediated RNA Silencing in Arabidopsis

“…Transcription of a terminator-less transgene is expected to generate improperly terminated, unpolyadenylated mRNAs that are known to be unstable (Drummond et al, 1985). However, we found that in the sde1-1 mutant background, substantial levels of mostly unpolyadenylated GUS transcripts were detected in the transformants harboring the terminator-less GUS construct (Figures 3B and 5).…”

Growing Xenopus oocytes have spare translational capacity.

“…The percentages of globin mRNA that remained stable in stage 4 relative to stage 6 oocytes were 12.8%:5.8%, 3.2%:2.5%, and 98%:75%, respectively, for the three experiments. Coincidentally, these experiments resolve another paradox present in the literature: Drummond et al (19) reported that the majority of globin mRNA injected into stage 6 oocytes is stable, in contrast to the study of Richter and Smith (4). Our current data show considerable variability among oocytes from different females, so that almost any percentage could be obtained, depending on the female donor used.…”

Localisation of RNAs into the Germ Plasm of Vitellogenic Xenopus Oocytes

“…Previously it has been reported that RNAs diffuse more slowly than proteins in oocytes [47]. Thus, if the injected RNAs and the Hermes protein diffuse independently one would expect Hermes, a relatively small protein, to spread faster through the oocyte than Cy5- nanos1 RNA.…”