Globin mRNA Species Containing Poly(A) Segments of Different Lengths

Abstract

Rabbit globin mRNA species containing poly(A) segments of different lengths were prepared by partial phosphorolysis of mRNA with Escherichia coli polynucleotide phosphorylase. By varying the salt concentration and the time of incubation of the phosphorolysis mixture, as well as performing oligo(dT)-cellulose chromatography at 22 "C and at 4 "C, globin mRNA preparations containing poly(A) segments of approximately 122, 95, 68, 39, 32,21, and 16 adenylate residues were obtained. It was found that the functional stability of the mRNA species containing 32 or more adenylate residues after injection into Xenopus luevis oocytes equaled that of the native globin mRNA. On the other hand, the functional stability of mRNA containing an average number of 21 adenylate residues was about 30% of the native mRNA, while that of mRNA containing 16 adenylate residues was as low as poly(A)-free globin mRNA.The existence of poly(A)-rich sequences at the 3'-end of most eukaryotic mRNAs is well established (for reviews see [l -31). The biological role of the poly(A) segment was the subject of many recent studies. Inhibition of post-transcriptional adenylation by the adenosine analogue cordycepin strongly reduces the flow of mRNA from the nucleus to the cytoplasm. This finding forms the basis for the suggestion that nuclear adenylation is an essential step in the biogenesis of mRNA or in its transport to the cytoplasm [4,5].Other findings, such as shortening of the poly(A) segment during the aging of mRNA [6,7] cytoplasmic adenylation [8,9], and the presence of a poly(A) segment in a viral message which is synthesized in the cytoplasm [lo], suggest an additional role for the poly(A) segment outside of the nucleus. Results reported from this and other laboratories have shown that the poly(A) segment of globin mRNA is not essential for its translation in cell-free systems [l 1 -161. However, preincubation in cell-free extracts decreases the template activity of poly(A)-free mRNA more than that of the native mRNA [16]. We have also reported that rabbit globin poly(A)-free mRNA is translated as efficiently as native mRNA during the Enzyme. Nucteosidediphosphate : polynucleotide nucleotidyltransferase (polynucleotide phosphorylase) (EC 2.7.7.8).first 45-min period of incubation in a Krebs-I1 ascites cell-free systems, but upon longer periods of incubation the rate of protein synthesis decreases more rapidly with the poly(A)-free mRNA than with the native mRNA [Ill. To overcome the time limitation of the cell-free system (which is active for only 90 min) we have used Xenopus luevis oocytes, which permit mRNA translation for some days. A pronounced difference between the functional stability of poly(A)-free and native globin mRNA was observed : whereas efficiency of translation of poly(A)-free mRNA in the oocytes is the same as that of the native globin mRNA during the first few hours after injection, 20-48 h later the efficiency of translation drops to about 10% of that of native globin mRNA [12]. Recently we have found that the drop in tem...

An analysis of the rate of metallothionein mRNA POLY(A)-shortening using RNA blot hybridization

“…Nudel et al (26) reported that the stability of globin mRNA molecules with a poly(A)-tail length of only 34 residues was indistinguishable from those molecules with longer poly(A)-tails when injected into Xenopus oocytes. This fact, taken together with the observed equal probability of decay of new or old mRNAs (with long or short poly(A)-tails respectively) (8) (10) who found that translation of poly(A)+ mRNAs in rabbit reticulocyte lysates could be selectively inhibited by addition of free poly(A).…”

“…This observation led to the suggestion that the length of poly(A) may be regulating mRNA stability (3,4) and artificially deadenylated mRNA is less stable when injected into Xenopus oocytes (5,6). A tail of 30 adenylate residues was found to be sufficient to stabilize globin mRNA (7). However, newly synthesized mRNA molecules, with long poly(A) tails were shown to be equally likely to be degraded as older mRNAs with short poly(A) tails (8).…”

Ribosome Concentration Contributes to Discrimination against Poly(A)− mRNA during Translation Initiation in Saccharomyces cerevisiae

“…The debate about the importance of poly(A) tails for mRNA translation reflects an attempt to combine experimental observations in systems in vitro including wheat germ extracts, rabbit reticulocyte lysates, and S. cerevisiae extracts (8 -11) and in living cells such as Xenopus laevis oocytes, HeLa cells, and S. cerevisiae (12)(13)(14)(15)(16)(17). In general, the in vitro studies demonstrated discrimination against the translation of poly(A)-deficient or poly(A) Ϫ input mRNAs; however, the extent of reducedtranslatabilitywasmodest,possiblyreflectinganextractdependent capacity for multiple rounds of translation initiation (i.e.…”

Globin Messenger RNA from Anaemic Rabbit Spleen Size of Its Polyadenylate Segment

“…We have shown that the poly(A) segment is required to ensure the stability of globin mRNA during its translation in Xenupus oocytes [15,20,21]. It might thus be supposed that, during the life of mRNA, the poly(A) stretch is progressively shortened up to a length which is no longer sufficient to protect messenger RNA against degradation [22]. ~3 1 .…”