Molecular composition of Ro small ribonucleoprotein complexes in human cells. Intracellular localization of the 60and 52-kD proteins

Ro small ribonucleoprotein complexes (RoRNPs) are thought to comprise several proteins, including the 60-kD Ro and the 52-kD Ro proteins, and several small RNAs, designated Y RNAs. Although RoRNPs are fairly ubiquitous in nature, their precise composition remains unknown, their function has been elusive, and their intracellular localization has been controver-sial. We have analyzed HeLa cell extracts by glycerol density gradient fractionation in order to determine the distribution of the individual protein and RNA components of RoRNPs. We found that 52-kD Ro was not detectable in an RNP complex with the 60-kD protein under a variety of conditions. Pretreat-ment of cell extracts with ribonuclease affected gradient migra-tion of the 60-kD but not the 52-kD protein, suggesting that the latter is not complexed with RNA. The migration of the hY RNAs in these gradients closely followed that of60-kD and not 52-kD Ro. Immunofluorescence analysis of two different cell lines with monospecific antibodies against 52- and 60-kD pro-teins strongly suggests that these two proteins are not present on overlapping sets of structures in vivo. We conclude that the 52-kD Ro protein is not a detectable component of the RoRNP complex under these conditions despite its reactivity with R