Translational activity and functional stability of human fibroblast beta 1 and beta 2 interferon mRNAs lacking 3'-terminal RNA sequences.

Abstract

Polyadenylylated mRNA was purified from poly(I) .cntdot. poly(C)- and cycloheximide-superinduced human fibroblast (FS-4) cultures. The mRNA was subjected to electrophoresis through an agarose/CH3HgOH gel, and human fibroblast .beta.1 and .beta.2 interferon mRNA were isolated. Each mRNA preparation was phosphorolyzed at 0.degree. C for 20 min by using a molar excess of polynucleotide phosphorylase [EC 2.7.7.8] to produce RNA lacking poly(A) and then incubated at 37.degree. C for varying lengths of time to allow the phosphorylase to further digest the deadenylylated RNA from the 3'' end in a processive and synchronous manner. Removal of the poly(A) (.ltoreq. 100 residues) and .apprxeq. 100 adjacent residues from human fibroblast .beta.1 interferon mRNA (native length, 900 residues, including a 3''-noncoding region of 203 residues) did not alter the translational activity or the functional stability of this mRNA in Xenopus laevis oocytes, whereas deletion of the poly(A) and .apprxeq. 200 adjacent residues decreased its translational efficiency. Removal of the poly(A) (.apprxeq. 200 residues) and .apprxeq. 200 adjacent residues from human fibroblast .beta.2 interferon mRNA (native length, 1300 residues) did not alter the translational activity or the functional stability of this molecule in oocytes. Thus, neither the poly(A) nor large segments of the 3''-noncoding region (which includes the hexanucleotide A-A-U-A-A-A sequence, at least in the case of .beta.1 mRNA) are required for the maintenance of the functional stability of human .beta.1 and .beta.2 interferon mRNA in X. laevis oocytes.