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

Abstract

The size of the polyadenylate segment of globin messenger RNA isolated from spleens of anaemic rabbits was estimated by comparison of its electrophoretic migration in polyacrylamide gels to that of synthetic poly(A) segments of known lengths. Conditions of enzymic degradation of mRNA with pancreatic ribonuclease and TI ribonuclease were carefully established in order to ensure complete degradation of the heteropolymeric part of mRNA without affecting the polyadenylate sequence. The poly(A) segments of spleen globin mRNA were found to be 25 -90 nucleotides long whilst those of peripheral blood reticulocytes from the same animals were only 10-30 residues long. Since spleen contains young erythroid cells and since anucleated blood reticulocytes constitute a statistically older population of the same cell line, these results support the idea that the poly(A) segment of mRNA shortens when the message ages.For the study of messenger RNA ageing it should be useful to isolate two messenger RNA populations of different ages coding for the same protein. For that purpose we isolated globin messenger RNA from spleens and from peripheral blood reticulocytes of phenylhydrazine-anaemied rabbits. In the case of haemolytic anaemia spleen becomes a major site of erythropoiesis and contains a globin mRNA population younger, on the average, than blood reticulocytes. These cells are relatively old, anucleated and constitute the penultimate step in the red cell line differentiation.In a previous paper [l] we described the isolation of globin messenger RNA from the spleens of anaemic rabbits. The method that was used included total RNA extraction from the tissue followed by poly(U)-Sepharose affinity chromatography and sucrose gradient centrifugations. However, from its relative heterogeneity in gel electrophoresis and its low biological activity in a protein-synthesizing system in vitro, it was obvious that this spleen mRNA preparation was less pure than the corresponding blood reticulocyte mRNA.In the same work we showed that these two mRNA preparations contain approximately the same proportion of poly(A). As spleen globin mRNA is prob- 203-208 (1970).Enzymes. Pancreatic ribonuclease (EC 3.1.4.22); ribonuclease TI (EC 3.1.4.8); SI nuclease (EC 3.1.4.21). ~~ ~~ably contaminated by non-messenger RNA, no clear conclusion could be reached as to the relative length of poly(A) in the two preparations.In the present work we directly measure, by gel electrophoresis, the lengths of the isolated poly(A) segments from spleen and reticulocyte globin mRNA and we can quantitatively compare the purities of these two globin mRNA preparations. METHODS Solutions UsedBuffer A : 0.02 M Tris, 0.3 M NaC1, 0.002 M EDTA brought to pH 7.5 with HCl.Electrophoresis buffer: 0.036 M Tris, 0.030 M NaH2P04, 0.001 M EDTA, 0.2% sodium dodecylsulphate, pH 7.6. Solution B. : 0.045 M sodium citrate, 0.45 M NaC1, 0.2 "/, sodium dodecylsulphate. Globin mRNA PrepamtionSpleen and blood reticulocyte globin messenger RNA were prepared as previously described (1). In order ...