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Structure of the gene encoding the rat T cell ecto-ADP-ribosyltransferase RT6
F A Haag1, G Kuhlenbäumer, F Koch-Nolte
1Department of Immunology, University Hospital Hamburg-Eppendorf, Germany.
Abstract:
Cellular functions, such as the cytolytic potential of CTLs, can be regulated by mono-ADP-ribosylation of target proteins. Recently, the T cell differentiation marker RT6 has been shown to possess mono-ADP-ribosyltransferase activity. Defects in RT6 expression coincide with increased susceptibility in animal models for insulin-dependent diabetes mellitus and other autoimmune diseases. We present an analysis of the rat RT6 gene, providing a basis for studying the regulation of this gene in T cells of normal and diabetes-prone rats. It is the first structural analysis of a mammalian mono-ADP-ribosyltransferase gene. The RT6 gene consists of eight exons spanning approximately 20 kb. The proximal four exons encode 5' untranslated region sequences and are found in multiple alternatively spliced variants. Exon 5 encodes the N-terminal signal sequence. An unusually large exon 7 encodes the entire native polypeptide. The final exon 8 encodes the C-terminal signal sequence for glycosylphosphatidylinositol anchor attachment and the 3' untranslated region. Two independent TATA box-containing promoters associated with exons 1 and 2 were identified, and their activity was verified in transient transfection assays. The distal promoter displays elements contained in the regulatory regions of T cell-specific genes, such as ets and ikaros. Analysis of RT6 transcripts showed that this promoter is the major one in adult rat spleen cells. The 3' end of the gene does not display alternative splicing. However, two polyadenylation signals are found in the 3' untranslated region.
Insights
The rat RT6 gene, crucial for T cell function and autoimmune disease regulation, has been structurally analyzed for the first time. This study details its gene structure, promoters, and alternative splicing, aiding future research into its role in diabetes.
Area of Science:
- Molecular Biology
- Immunology
- Genetics
Background:
- Cellular functions, including T cell cytolytic activity, are regulated by mono-ADP-ribosylation.
- The T cell marker RT6 exhibits mono-ADP-ribosyltransferase activity, and its deficiency is linked to autoimmune diseases.
Purpose of the Study:
- To perform the first structural analysis of the mammalian RT6 gene.
- To provide a foundation for studying RT6 gene regulation in normal and diabetes-prone rats.
Main Methods:
- Gene structure analysis of the rat RT6 gene.
- Identification and functional verification of gene promoters using transient transfection assays.
- Analysis of RT6 gene transcripts and polyadenylation signals.
Main Results:
- The RT6 gene comprises eight exons over ~20 kb, with alternative splicing in the 5' untranslated region.
- Two distinct promoters were identified, with the distal promoter being dominant in spleen cells and containing T cell-specific regulatory elements.
- The gene features a large exon 7 encoding the polypeptide and exon 8 for GPI anchor attachment, with two polyadenylation signals in the 3' UTR.
Conclusions:
- This structural characterization of the rat RT6 gene is essential for understanding its regulation in T cells.
- The identified promoters and regulatory elements offer insights into the control of RT6 expression.
- Further research can now investigate the role of RT6 in autoimmune conditions like diabetes.