Purification by benzoylated cellulose chromatography of translatable messenger ribonucleic acid lacking polyadenylate.

A population of mRNA that does not bind to oligo(dT)-cellulose (operationally defined as poly(A)-mRNA) has been purified 50-fold by benzoylated cellulose chromatography.The nominal poly(A)-mRNA is authentic mRNA on the basis of its labeling in the absence of rRNA synthesis, its sedimentation pattern, its release from polysomes by EDTA, and its activity as template in a heterologous cell-free translation system.Only 40 to 50% of rapidly labeled RNA originally retained on benzoylated cellulose is polyadenylated as judged by its subsequent retention on oligo(dT)-cellulose.Similarly, the amount of nominal poly(A)-mRNA and poly(A)+ mRNA appears to be equivalent on the basis of labeling studies and translation in the rabbit reticulocyte system.Chromatography of renal poly-soma1 RNA labeled under conditions that suppress cytoplasmic accumulation of newly synthesized rRNA showed approximately 30 to 35% of labeled mRNA did not bind to either benzoylated cellulose or oligo(dT)cellulose.Quantitation of rRNA-specific, methyl-labeled dinucleotides in purified nominal poly(A)-mRNA showed 98% of rRNA is removed by these chromatographic procedures.Purification of biologically active poly(A)-mRNA by benzoylated cellulose chromatography should be useful in further delineating the regulatory role of poly(A)-mRNA and in investigating the metabolism of mRNA in the cytoplasm of mammalian cells and organs.Although the studies of mammalian mRNA have relied primarily on analysis of mRNA isolated using its 3'-OH terminal polyadenylate as a marker (1, 2), a substantial fraction (-30%) of rapidly labeled polysomal mRNA in exponentially growing cultured mouse L-cells and HeLa cells lacks poly (A),' judged by its inability to bind to oligo(dT)-cellulose (3-5 ) .Translatable mRNAs for several proteins, including actin and protamine, are present in both the polyadenylated and the nonadenylated state in eukaryotic cells (6-10).Thus, analysis of only polyadenylated mRNA (poly(A)+ mRNA) may not provide a sufficiently thorough description of mRNA metabolism.For example, because the possible deadenylation of poly(A)' mRNA would result in biologically active mRNAs that no longer contain poly(A), the existence of poly(A)-lacking mRNA (poly(A)-mRNA) raises doubts of estimates of