Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


A Novel Splicing Regulatory Mechanism in Generating a Truncated Human Death Receptor 3 (DR3) Gene Product That Contributes to the Pathogenesis of Rheumatoid Arthritis.

Mizuhara2,  Masaru, Hashiramoto3,  Akira, Yoshida1,  Kohsuke, Shiozawa4,  Shunichi

Department of Biophysics, Kobe University Graduate School of Health Science, Kobe, Hyogo, Japan
Department of Biophysics, Kobe University Graduate School of Health Science, Kobe, Japan
Department of Biophysics, Kobe University Graduate School of Health Science/Department of Medicine, Kobe University Graduate School of Medicine/The Center for Rheumatic Diseases, Kobe University Hospital, Kobe, Hyogo, Japan
Department of Biophysics, Kobe University Graduate School of Health Science/Department of Medicine, Kobe University Graduate School of Medicine/The Center for Rheumatic Diseases, Kobe University Hospital, Kobe, Japan

Purpose:

Death Receptor 3 (DR3), a member of tumor necrosis factor receptor superfamily, transmits both apoptosis and NFkB activation signalings. We previously reported a new DR3 haplotype containing 4 single-nucleotide polymorphisms (SNPs) and one 14-nucleotide deletion within exon 5 and intron 5 (GenBank accession Nos. AB051850 to DR3 and AB051851), which resulted in insertion of a portion of intron 5 into the coding sequence to generate a premature stop codon. Based on these mutations, we identified a mutant type of DR3 gene (mtDR3) encoding a truncated DR3 lacking tramsmembrane domain and death domain as the candidate disease gene for Japanese-Korean patients with rheumatoid arthritis (RA). In this study, we show a specific binding of splicing regulatory proteins on intron 5 in the mtDR3 gene, which leads to generation of a truncated DR3 lacking death domain molecule contributing to the pathogenesis of RA.

Methods:

The wild type (wt) exon 2, wt intron 5 and mutant intron 5 pre-mRNA were synthesized from wtDR3 and mtDR3 genes, respectively, using PCR and DNA transcription and were incubated with nuclear proteins extracted from jurkat cells. The nuclear protein bound to intron 5 pre-mRNA was collected, separated on SDS-Polyacrylamide gels and analysed by silver staining, mass spectrometric and western blotting.

Results:

We previously identified that the mutation of DR3 gene, the polymorphism is: d: g.2590A>T (rs3138155) from the first base of ATG, enhances insertion of a portion of intron 5. As to the cause of exon insertion at intron 5, we have detected 3 nuclear proteins with molecular weight, 100kDa, 70kDa and 60kDa which bound to the mutant type intron 5, but not to wild type intron 5, of DR3 mRNA under silver staining. Mass spectrometric and western blotting identified them as splicing factors, proline- and glutamine-rich (SFPQ), heterogeneous nuclear ribonucleoprotein L (hnRNP L) and non-POU domain-containing octamer-binding protein (NONO), respectively.

Conclusion:

As to the cause of exon insertion at intron 5, the truncated DR3 lacking death domain molecule contributes to the pathogenesis of RA. We have identified 3 splicing regulatory proteins, SFPQ, hnRNP L and NONO, that specifically bound to the intron 5 mtDR3 to generate a truncated DR3 gene product.

To cite this abstract, please use the following information:
Mizuhara, Masaru, Hashiramoto, Akira, Yoshida, Kohsuke, Shiozawa, Shunichi; A Novel Splicing Regulatory Mechanism in Generating a Truncated Human Death Receptor 3 (DR3) Gene Product That Contributes to the Pathogenesis of Rheumatoid Arthritis. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :348
DOI: 10.1002/art.28117

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