PURIFICATION OF ANTIGENICALLY INTACT RO RIBONUCLEOPROTEINS - BIOCHEMICAL AND IMMUNOLOGICAL EVIDENCE THAT THE 52-KD PROTEIN IS NOT A RO PROTEIN

Anti-Re sera immunoprecipitate Ro ribonucleoproteins (RNPs) from human cell extracts. Ro RNPs are biochemically heterogeneous particles whos e functions are unknown and whose exact composition remains controvers ial. In addition to 60-kD Ro and to La proteins, a 52-kD polypeptide ( p52) has been proposed to be a stable component of the Ro RNPs. To con firm the immunological studies supporting this hypothesis, we have bio chemically purified Ro RNPs from HeLa cells using non-denaturing condi tions. Ro RNPs segregated into three distinct populations, one of whic h only contained hY5 RNA (Ro(hY5) RNPs). No p52 co-purified with Ro RN Ps. Despite the absence of p52, purified Ro RNPs had biochemical and i mmunological properties identical to those of unfractionated Ro RNPs. Many anti-Re sera only recognize p52 in immunoblots, and are said to b e monospecific anti-p52. Preincubation with purified Ro(hY5) RNPs (fre e of p52) of all human anti-Re (including so-called monospecific anti- p52) sera abolished their capacity to immunoprecipitate Ro RNPs from u nfractionated HeLa cell extracts. Conversely, preincubation of anti-Re sera with purified p52 protein specifically inhibited recognition of p52 in immunoblots, but did not interfere with immunoprecipitation of Ro RNPs. Our data demonstrate that anti-p52 antibodies do not target i ntact Ro RNPs, nor do they target the native 60-kD Ro protein. Contrar y to previous reports, p52 protein is not a stable component of antige nically intact Ro RNPs.