ALTERATIONS IN THE SIZE OF THE POLY(A) SEGMENT IN NEWLY-SYNTHESIZED MESSENGER RNA OF MOUSE SARCOMA 180 ASCITES CELLS GEORGE BRAWERMAN Department of Biochemistry and Pharmacology, Tufts University School of Medicine, Boston, Mass. 02111, U.S.A. (Received 7 March, 1973) ABSTRACT. The size of the poly(A) segment present at the 3'-end of mRNA molecules decreases gradually after entry of the mRNA into the cytoplasm. The size reduction seems to occur equally in free and polysome-bound mRNA. It is not affected by conditions that inhibit protein synthesis, such as treatment with cycloheximide and amino acid deprivation. The latter condition leads to a substantial increase in the size of nuclear poly(A). The rate of poly(A) cleavage could determine the length of time a mRNA molecule remains functional in the cytoplasm. I. INTRODUCTION Most mammalian messenger RNA has been shown to contain a large polyadenylate sequence, localized at the 3'-end of the RNA molecules [1-5]. The same sequence is present in the nucle- oplasmic RNA molecules presumed to be precursors of cytoplasmic mRNA [3-5]. Current evidence suggests that poly(A) synthesis takes place by sequential addition of adenylate residues to the newly-synthesized nuclear RNA molecules [5, 6]. The size of the poly(A) segment is subject to considerable variation. The newly-synthesized poly(A) of mouse sarcoma 180 ascites cells appears to contain about 200 nucleotides, while in cytoplasmic mRNA it is about 170 nucleotides long after a 2-hr labeling period [5]. Segments containing about 50 residues have been recovered from rabbit globin mRNA labeled for a long period of time [1], and stretches of only 5-7 residues have been reported at the 3'-end of the unlabeled globin RNA [7]. The average size of the poly(A) seg- ments from steady-state labeled mouse L-cells has been shown to be substantially smaller than that of 1-hr labeled cells, and their size distribution more heterogeneous [8]. It is possible, there- fore, that the poly(A) segment undergoes a gradual decrease in size in the course of mRNA function. The identification of the cellular processes concerned with the apparent reduction in poly(A) size could provide information on the role of the poly(A) segment. It is conceivable, for instance, that the transport of the mRNA from nucleus to cytoplasm might involve the cleavage of a group of adenylate residues. A cleavage could also take place to activate the mRNA and permit its initial interaction with ribosomes, and a gradual reduction in poly(A) size might occur as the mRNA undergoes rounds of translation. In order to investigate these possibilities, the poly(A) segments in newly-formed RNA from nuclei, free cytoplasmic particles and polysomes were com- pared, using electrophoretic mobility in polyacrylamide gel as a measure of size. The effect of con- Molecular Biology Reports 1 (1973) 7-13. All Rights Reserved Copyright ?9 1973 by D. Reidel Publishing Company, Dordrecht-Holland
