Splicing en el MHC de clase III caracterización y expresión de las isoformas del gen NFkBIL1estudio de su relación con artritis reumatoide

Zusammenfassung

A major discovery in the post-genomic era is that the number of proteins in a human proteome is by no means equivalent to the number of genes in the genome. Alternative splicing (AS) is one of the mechanisms that increases transcriptome and proteome diversification by generating multiple mRNA products from a single gene. The human major histocompatibility complex (MHC) is located on the short arm of chromosome 6 and is divided into the class I, extended class I, class II, extended class II and class III regions. The MHC contributes to a large number of immune-related disorders including insulin dependent diabetes mellitus, rheumatoid arthritis (RA) and common variable immunodeficiency. The human NF-¿B inhibitor-like protein 1 (NF¿BIL1) gene, also known as I¿BL, is located at the telomeric end of the Major Histocompatibility Complex (MHC) class III region on chromosome 6. It encodes a protein resembling members of the I¿B protein family with ankyrin repeat sequences that may interact with members of the nuclear factor ¿B (NF- ¿B)/Rel family. NF-¿B controls many genes involved in inflammation so it is not surprising that NF-¿B is found to be chronically active in many inflammatory diseases. Two mRNA variants of NF¿BIL1 were previously described and we have identified two new isoforms in humans. We have characterised NF¿BIL1 splicing isoforms in different species as well. In this study, we analysed NF¿BIL1 isoforms expression levels in different human cell lines and tissues and we investigated the possible role of this gene in the development of RA by studying expression levels of each isoform in RA patients and controls. For precise analyses of NF¿BIL1 expression, mRNA levels of the four isoforms identified were measured by real-time RT-PCR analysis. NF¿BIL1 mRNA isoforms present different expression levels in all samples studied, being the canonical isoform NF¿BIL1 the most expresed in all cases. To describe the distribution of all NF¿BIL1 protein isoforms, localisation experiments with confocal miscroscopy were performed. Transient expression of NF¿BIL1 proteins showed that all of them were targeted to the nucleus, specifically in nuclear speckles, suggesting a possible role in the regulation of splicing. We also quantified NF¿BIL1 mRNA isoforms in blood samples of healthy controls and RA patients by real-time RT-PCR analysis. Different expression levels of NF¿BIL1 isoforms were also found between RA patients and controls, showing a clear overexpression of each isoform in RA patients. These data suggest that NF¿BIL1 variants may be related with the regulation of splicing and the overexpression of each isoform in RA patients lend support to a role of this gene in the pathogenesis of RA. We questioned whether the level of DNA methylation in NF¿BIL1 promoter region may be relevant to NF¿BIL1 expression and may be a possible contributory factor in the inflammatory pathogenesis of RA. A detailed methylation analysis of the CpG sites in samples of controls and RA patients was performed but our results showed that the methylation pattern did not change between cases and controls, suggesting that mechanisms other than methylation are involved in regulation of NF¿BIL1 expression.