Analysis of the molecular composition of Ro ribonucleoprotein complexes - Identification of novel Y RNA-binding proteins

Human Ro ribonucleoproteins (RNPs) are composed of one of the four small Y RNAs and at least two proteins, Ro60 and La; association of additional prot eins including the Ro52 protein and calreticulin has been suggested, but cl ear-cut evidence is still lacking. Partial purification of Ro RNPs from HeL a S100 extracts allowed characterization of several subpopulations of Ro RN Ps with estimated molecular masses of between 150 and 550 kDa. The majority of these complexes contained Ro66 and La, whereas only a small proportion of Ro52 appeared to be associated with Ro RNPs. To identify novel Y RNA-ass ociated proteins in vitro, binding of cytoplasmic proteins to biotinylated Y RNAs was investigated. In these reconstitution experiments, several prote ins with estimated molecular masses of 80, 68, 65, 62, 60 and 53 kDa, the l atter two being immunologically distinct from Ro60 and Ro52, respectively, appeared to bind specifically to Y RNAs. Furthermore, autoantibodies to the se proteins were found in sera from patients with systemic lupus erythemato sus. The proteins bound preferentially to Y1 and Y3 RNA but, with the excep tion of the 53-kDa protein, only weakly to Y4 RNA and not at all to Y5 RNA. Coprecipitation of the 80, 68, 65, and 53-kDa proteins by antibodies to Ro 60 and La was observed, suggesting that at least a proportion of the novel proteins may reside on the same particles as La and/or Ro60. Finally, the b inding sites for these proteins on Y1 RNA were clearly distinct from the Ro 60-binding site involving a portion of the large central loop 2, which was found to be indispensable for binding of the 80, 68, 65 and 53-kDa proteins , as well as the stem 3-loop 3 and stem 2-loop 1 regions. Interestingly, tr uncation of the La-binding site resulted in decreased binding of the novel proteins (but not of Ro60), indicating La to be required for efficient asso ciation. Taken together, these results suggest the existence of further sub populations of Ro RNPs or Y RNPs, consistent with the heterogeneous charact eristics observed for these particles in the biochemical fractionation expe riments.