Phosphorylation in vivo of Proteins Associated with Heterogenous Nuclear RNA in HeLa Cell Nuclei

As a step towards understanding the role of nuclear ribonucleoprotein particles (hnRNA · proteins) in the nucleocytoplasmic transfer of heterogenous nuclear RNA sequences destined to become the cytoplasmic messenger RNA, the phosphorylation in vivo of the protein moiety of these hnRNA · proteins from HeLa cells has been studied. After exposure of HeLa cells to [ 32 P]orthophosphate in vivo , purified hnRNA · proteins were found to contain radioactivity covalently bound to proteins in the form of serine and threonine phosphate esters. The association of these phosphoproteins with hnRNA was demonstrated by sedimentation analysis in sucrose gradients, isopycnic banding in cesium chloride density gradients and chromatography on oligo(dT)‐cellulose. The pattern of radioactive phosphorylated species has been analysed by electrophoresis on sodium dodecylsulphate/polyacrylamide slab gels followed by autoradiography. Although some hottrichloroacetic‐acid‐resistant 32 P radioactivity was present throughout the gel, the highest amount of label was found associated with four discrete bands of molecular weights 28000, 30000, 37000 and 52000. Incorporation of 32 P into hnRNA · proteins could be detected after labeling periods as short as 15min, the same species being labeled after 1 and 24‐h labeling times. Although all the above species appear to be present in hnRNA · proteins of different size classes, as resolved by sedimentation through sucrose gradients, their relative proportion is not reproducibly constant. Exposure of hnRNA · proteins to conditions of increasing ionic strength led to the ready dissociation of the 28000‐ M r species at variance with other labeled species which appeared to be more firmly bound to hnRNA than the bulk of proteins. Upon treatment with pancreatic and T 1 ribonucleases, the 28000‐ M r species was also preferentially released while the 37000‐ M r one was the main phosphorylated species remaining associated with core structures containing an RNA component resistant to nucleases which is now under further characterization.