52-kD SS-A/Ro: genomic structure and identification of an alternatively spliced transcript encoding a novel leucine zipper-minus autoantigen expressed in fetal and adult heart
Chan, E.K.; Di Donato, F.; Hamel, J.C.; Tseng, C.E.; Buyon, J.P.
Journal of Experimental Medicine 182(4): 983-992
The 52-kD SS-A/Ro protein is one of the antigenic targets strongly associated with the autoimmune response in mothers whose children have manifestations of neonatal lupus. In addition to the cDNA clone we previously reported for the full-length 52-kD SS-A/Ro protein, an interesting MOLT-4 cDNA clone, p52-2, was found to have an internal deletion of 231 nucleotides including the domain encoding the leucine zipper motif. To further investigate the nature of this deletion, genomic DNA clones were isolated from a lambda-FIXII library. The complete gene for the full-length 52-kD protein (alpha form, 52-alpha) spans 10 kb of DNA and is composed of seven exons. Exon 1 contains only the 5' untranslated sequence, while the translation initiation codon is located 3 kb downstream in exon 2, which also encodes the three zinc finger motifs. Exon 4 encodes amino acids 168-245, including the coiled coil/leucine zipper domain. Exon 7 is the longest and encodes the rfp-like domain and the 3' untranslated region. The cDNA p52-2 can now be accounted for as a product of alternative messenger RNA (mRNA) derived from the splicing of exon 3 to exon 5, skipping exon 4, which results in a smaller protein (52-beta) with a predicted molecular weight of 45,000. An initial approach to identifying this alternatively spliced form in the human heart used a ribonuclease protection assay. Using an PNA probe corresponding to bases 674-964 of the full-length cDNA, two protected mRNA fragments were identified, a 290-bp fragment corresponding to expression of 52ot and a smaller fragment of 144 bp, the predicted size of 52-beta. Using reverse transcription followed by polymerase chain reaction, cDNAs from a 16-wk fetal heart, 24-wk heart, and adult heart were amplified with primers flanking exon 4. Two polymerase chain reaction products were observed in each tissue, one 1.0 kb likely representing 52-alpha and a second 0.78 kb, consistent with 52-beta. The 0.78-kb fragment identified in the 16-wk heart was cloned, and DNA sequencing confirmed the 52-beta type. Immunoprecipitation of in vitro-translated 35S-labeled 52-beta form was performed to evaluate the antigenicity of this novel form of 52-kD SS-A/ Ro.26 (87%) of 30 sera tested from mothers whose children were known to have neonatal lupus immunoprecipitated the 52-beta form. In summary, we herein describe an alternatively spliced mRNA encoding a novel 52 SS-A/Po autoantigen expressed in a variety of tissues including the fetal heart. Precognition of this 52-beta isoform suggests that antigenicity of the 52-kD SS-A/Ro protein can be independent of the putative leucine zipper domain. These findings may further elucidate the cellular function of the 52-kD SS-A/Po antigen and its role in the development of an autoimmune disease and subsequent injury to fetal tissue.