Isolation of messenger ribonucleoproteins from mammalian cells

EDTA-dissociated polysomes from normally growing and adenovirus-infected KB cells were fractionated by affinity chromatography on oligo(dT)-cellulose into an unadsorbed fraction containing the ribosomal subunits and a second fraction which bound to the adsorbent. The latter fraction, recovered from the oligo(dT)-cellulose by elution with formamide in a buffer containing 0.2 m-NaCl, was shown to contain messenger RNA together with protein. Evidence was obtained that a substantial part (50%) of ³⁵S-labelled protein was associated with the mRNA when eluted from the oligo(dT)-cellulose, and that up to 75% of the labelled protein was mRNA-associated after dilution of the eluted material in buffer not containing formamide. As judged from the characteristic gel electrophoresis pattern of adenovirus-specific mRNA derived from such messenger ribonucleoprotein complexes, the mRNA remained intact during the isolation procedure. Analysis of the fraction containing the majority of the messenger ribonucleoprotein complexes on sodium dodecyl sulfate-polyacrylamide gels showed a specific pattern of labelled polypeptides, which in the case of material from uninfected cells consisted of four major labelled polypeptides; approximate molecular weights 56,000, 68,000, 78,000 and 130,000. When the material from the adenovirus infected cells was analysed a set of four polypeptides, which migrated identically to those of the uninfected cells, was found. However, in addition to this apparently common set of polypeptides, the material from the infected cells, harvested late in the infectious cycle, contained one extra polypeptide with an approximate molecular weight of 110,000.