POLYNUCLEOTIDE PHOSPHORYLASE AS A PROBE FOR THE REGULATORY FUNCTION OF THE 3′-OH REGION OF mRNA AND VIRAL RNA IN TRANSLATION

Abstract

A method has been developed for the removal of the 3′-poly(A) tail from mRNA chains. It is based on synchronous processive phosphorolysis of mRNA using a molar excess of polynucleotide phosphorylase at 0°. This method enabled to determine the location, length and cytoplasmic role of the poly(A) tail of globin mRNA. When injected into Xenopus oocytes, deadenylated rabbit globin mRNA was shown to be translated for a relative short period and then rapidly degraded. Native poly(A)-containing mRNA, however, is considerably more stable in the same conditions and is translated for extended periods of time. The poly(A) stretch must contain a minimal number of about 30 adenylate residues to ensure its protective function. The increased stability conferred upon globin mRNA by the presence of the poly(A) tail is not a general phenomenon. Thus the translational stability in oocytes of poly(A)-containing mengovirus RNA is indistinguishable from that which is poly(A)-deficient. A regulatory role for the heterogenous 3′-end sequence in the translation of carnation mottle virus (CarMV) RNA was revealed. CarMV RNA seems to be polycistronic as it is translated in vitro into three discrete polypeptide chains (CP-I CP-II and CP-III) of which CP-II is identical with the viral coat protein. A selective reduction in the translation efficiency of CP-I could be elicited by controlled phosphorolysis of the 3′-terminus of CarMV RNA chains. This was paralleled with a gradual loss of infectivity. On the other hand phosphorolysis of only 5-10 nucleoside residues from the 3′-terminus of tobacco mosaic virus RNA eliminated its infectivity in tobacco leaves.