Two‐Dimensional Analysis of Proteins Associated with Heterogenous Nuclear RNA in Various Animal Cell Lines

Abstract

The protein complement of heterogenous nuclear RNA · protein particles from human HeLA, mouse L and Chinese hamster (CHO) cells has been analysed by two‐dimensional gel electrophoresis using the two techniques described by O'Farrell [ J. Biol. Chem. (1975) 250 , 4007–4021 and Cell (1977) 72 , 1133–1142]. Over a hundred individual spots have been reproducibly detected both in stained gel and in autoradiographs of proteins labeled with L ‐[ 35 S]methionine. Large similarities, especially in the 25000–40000 M r cluster of basic protein, were found among these three mammalian species. As far as phosphoproteins are concerned, it was observed that the bands already described by one‐dimensional gels [ Eur. J. Biochem. (1978) 86 , 301–310] with M r values of 28000, 30000, 37000 and 52000 are resolved into about 15 individual spots, suggesting a corresponding number of distinct states of phosphorylation. It was also clearly demonstrated that phosphoproteins are unrelated to the major basic protein species. Particles of different size classes were analysed with respect to their content of individual proteins, both non‐phosphorylated and phosphorylated. The most salient feature observed was that phosphoproteins become progressively more abundant with particles of increasing size. This raises the possibility that at least some of these phosphoproteins might belong to a nuclear structure to which hnRNA is normally bound.