title: Human SS-B/LA autoantigen contains a covalent protein-RNA linkage.
abstract: The chemical nature of association of RNA in immunoprecipitates of human SS-B/La ribonucleoprotein, an autoantigen expressed in various autoimmune disorders, was investigated. A fraction of RNA associated with SS-B/La immunoprecipitates was readily dissociated by SDS-polyacrylamide gel electrophoresis, yielding four main subfractions, R1-4, with chain lengths in the range of 90-130 nucleotides (R4), 140-175 nucleotides (R2 and R3) and above 200 nucleotides (R1). Moreover, the immunoreactive protein component, migrating with a molecular mass of 49 kDa, contained a very tightly bound RNA co-migrating with the protein unless the protein was proteolytically degraded. Most of the RNA molecules in this fraction, represented by about 20 components, had a free 3'-terminus but a blocked 5'-terminus and showed chain lengths between 10 and 125 nucleotides. After pretreatment with alkaline phosphatase and a mixture of ribonucleases T1 + T2 + A, adenosine 3',5'-biphosphate (pAp) was liberated by phosphodiesterase (Crotalus durissus) as the blocked 5'-end of the RNA. The chemical nature of the blockage was revealed after alternative treatment of the protein-pAp component with phosphodiesterase or nuclease S7 followed by acid hydrolysis and phosphoamino acid analysis which showed that a threonine residue must be directly involved in the RNA-protein linkage of 49 kDa SS/La antigen, indicating the presence of a covalent threonine-pAp bond.
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D001324:Autoantigens / Q000378:metabolism*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014157:Transcription Factors; D000097585:SS-B Antigen
chemical_list: D000469:Alkaline Phosphatase; D001324:Autoantigens; D019286:Endopeptidase K; D010450:Endopeptidases; D010727:Phosphoric Diester Hydrolases; D012313:RNA; D012260:Ribonucleases; D012261:Ribonucleoproteins; D013912:Threonine; D014157:Transcription Factors; D000097585:SS-B Antigen