Translational Properties of Rabbit Globin mRNA after Specific Removal of Poly(A) with Ribonuclease H

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Abstract
Highly purified RNase H (RNA.cntdot.DNA hybrid ribonucleotidohydrolase, EC 3.1.4.34) from calf thymus was used to specifically remove the poly(A) sequences of purified rabbit globin mRNA after its hybridization with poly(dT). The deadenylylated globin mRNA was repurified by a one-step procedure including a nitrocellulose column. The poly(A) size and the content of unmodified mRNA were determined by hybridization with [3H]-poly(U); the RNase H digestion method effectively removed this terminal poly(A) sequence. No difference in activity was found between mRNA with and without poly(A) to initiate, elongate, terminate and release newly synthesized globin chains in exogenous-mRNA-dependent, cell-free protein-synthesizing systems from wheat embryo, [mouse] ascites Krebs II cells and rat liver. Poly(A)-free globin mRNA competed with the same efficiency as authentic globin mRNA against chick ovalbumin mRNA when translated under total mRNA saturation conditions. The 3''-terminal poly(A) sequence is not necessary to maintain the translationally active secondary and tertiary configuration of the globin mRNA molecule. Preincubation of intact and deadenylylated globin mRNA in the Krebs II ascites translational system indicates that the presence of the poly(A) sequence may stabilize the translationally active mRNA molecule.

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