Control of Enzymic Hydrolysis of Polyadenylate Segment of Messenger RNA: Role of Polyadenylate‐Associated Proteins

Abstract

The role of poly(A)‐associated proteins in the breakdown of poly(A) sequences in both mammalian polyribosomes and in isolated poly(A) · protein complexes has been studied on an enzymic level. Two nucleases (alkaline exoribonuclease and endoribonuclease IV; both isolated from eukaryotic tissue), which preferentially hydrolyze poly(A) sequences, have been applied to determine the susceptibility of poly(A) in dependence on the presence of poly(A) · protein(s). Polysomes, isolated from L5178y mouse lymphoma cells, do not contain endogenous poly(A) nuclease activity. The poly(A) segment in polysomes is hydrolyzed by the exoribonuclease, irrespective of the preincubation conditions used. Pretreatment of the polysomes with 1 M urea increases the rate of hydrolysis considerably. In contrast to susceptibility of poly(A) towards the exoribonuclease, the endoribonuclease IV does not hydrolyze poly(A) in mammalian polyribosomes under low salt conditions (140 mM KCl). However pretreatment of the polysomes with 1 M urea renders the poly(A) susceptible to endoribonuclease IV. Similar results were obtained using poly(A) · protein complex, which has been isolated from mRNA · protein by ribonuclease digestion; two polypeptide species were found to be present in this complex, one with a molecular weight of 79000 and a second one with a molecular weight of 51000. While the poly(A) segment is hydrolyzed by exoribonuclease both after low and 1 M urea salt pretreatment, the homopolymer can be degraded by endoribonuclease IV only after a pretreatment with urea. In control experiments it was established that under incubation conditions of 1 M urea a poly(A)‐associated protein is dissociated from the homoribopolymer. Therefore we conclude that the susceptibility of poly(A) to nucleolytic enzymes is determined by the interaction with a poly(A)‐associated protein. Copyright © 1978, Wiley Blackwell. All rights reserved