ABSTRACT
The Ro complex is composed of three ribonucleoproteins of 60, 54, and 52 kDa. The Ro60 is associated with the 1-5 hYRNAs. Autoantibodies against Ro are found in sera from lupus patients. Lupus frequently presents in female patients during childbearing years and transplacental passage of maternal anti-Ro/La autoantibodies has been implicated in triggering neonatal lupus and congenital heart block (CHB). Since expression of Ro ribonucleoproteins occurs before the onset of CHB, epitope spreading of Ro hYRNAs could arise during cardiac ontogeny, thus maternal autoantibodies would damage a previously developed AV node. To explore the cardiac ontogeny of Ro hYRNAs, embryonic and adult tissues were obtained from legal autopsies. In situ hybridization using oligonucleotides for hY1, hY3, hY4, hY5, and Ro60 was performed on sections of cardiac tissue; their corresponding cDNAs were amplified by RT-PCR. The principal results were as follows: expression of hY4/5RNAs was greater in fetal tissues from 8-12 weeks of development. The subcellular distribution of hYRNAs in embryonic tissues was nucleocytoplasmic, whereas in the adult heart it was cytoplasmic. In conclusion, hYRNAs expression occurs during early cardiac development.
Subject(s)
Atrioventricular Node/chemistry , Autoantigens/genetics , RNA, Ribosomal, 5S/analysis , RNA, Small Cytoplasmic , Ribonucleoproteins/genetics , Adult , Atrioventricular Node/embryology , Fetus/chemistry , Gene Expression Regulation, Developmental , Humans , In Situ Hybridization , Oligonucleotide Probes , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Ro particles are conserved molecules that contain a YRNA and various Ro proteins, which are recognized by autoimmune sera from patients with lupus erythematosus or Sjögren's syndrome. The Ro60 ribonucleoprotein (RNP) forms complexes with certain 5S rRNAs, in such a manner that Ro60 could participate in the control of 5S rRNA production. The present studies were carried out to explore the interaction of Ro components, and to address the question whether Ro60 RNP binds simultaneously 5S rRNA and hYRNA. Anti-Ro60 antibodies were used to immunoprecipitate the RNA. Immunoprecipitates were reverse transcribed with specific oligonucleotides and the resulting cDNAs from 5S and hY4 were amplified by PCR. We found that 5S rRNA is complexed with hY4 and hY5 RNAs by means of the Ro60 RNP. Moreover, by in situ hybridization assays we were able to demonstrate that these molecules have a similar nuclear distribution. According to these results, it seems reasonable to assume that the Ro60 protein could be involved in ribosome assembly.
Subject(s)
Autoantigens/metabolism , RNA, Ribosomal, 5S/metabolism , RNA, Small Cytoplasmic/metabolism , Ribonucleoproteins/metabolism , Antibodies, Monoclonal/isolation & purification , Autoantigens/immunology , Autoantigens/isolation & purification , Cell Nucleus/chemistry , Humans , Lupus Erythematosus, Cutaneous/blood , Lupus Erythematosus, Cutaneous/immunology , Macromolecular Substances , Molecular Weight , Nucleosomes/metabolism , Precipitin Tests , Protein Binding , RNA, Ribosomal, 5S/immunology , RNA, Small Cytoplasmic/immunology , RNA, Transfer , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleoproteins/immunology , Ribonucleoproteins/isolation & purificationABSTRACT
Centromeres are critical structures in cell division, and CENP-B is the most important protein of the centromeric complex recognized by autoantibodies from patients with scleroderma. Our major aim was to demonstrate whether CENP-B is a conserved protein along the phylogenic scale including the higher plants. Vegetal and human cell proteins were extracted from Phaseolus vulgaris and HEp-2 cells and were characterized by PAGE, Western blot, and human autoimmune sera containing anti-CENP-B autoantibodies. The aminoterminus of the gene encoding for CENP-B from HEp-2 cells and Phaseolus vulgaris was isolated by reverse transcriptase-PCR using complementary oligonucleotides to the human CENP-B gene. Also, in situ hybridization was performed on vegetal tissues and HEp-2 cells using human CENP-B box probes. Our main results were as follows: 1) Autoimmune sera were reactive to a vegetal protein of 80 kDa. 2) Affinity-purified anti-CENP-B antibodies recognized a protein from Phaseolus vulgaris with molecular mass similar to that found in human cells. Vegetal and HEp-2 cells CENP-B proteins were immunologically identical. 3) Using RT-PCR, we were able to amplify a cDNA encoding for the aminoterminus domain of CENP-B from Phaseolus vulgaris that had the same molecular behaviour as the cDNA from HEp-2 cells. 4) Complementary oligonucleotides for human CENP-B box hybridized a DNA sequence from Phaseolus vulgaris. In conclusion, CENP-B protein is a conserved protein along the phylogenic scale from humans to higher plants.
