Estudio comparativo del splicing alternativo del gen NCR3 en diferentes especies de mamíferos y sus posibles implicaciones funcionales

Abstract

Alternative splicing (AS) is a major source of transcriptome and proteome diversity in higher eukaryotes allowing the generation of several structurally and functionally distinct mRNAs and protein isoforms from a single gene. Differential AS variants expression has been implicated in tissue and cellular differentiation, which in an evolutionary context could account for the phenotypic divergence of mammals that share a common repertoire of genes. On the other hand, the miss-regulation of AS is one of the mayor sources of human disease. However, there are not many detailed comparative analyses showing the alternative splice forms generated from particular genes among different organisms. The work presented here is a deep study of the splice variants expressed by the “Natural Cytotoxicity Receptor 3” (NCR3) gene and its comparative analysis among 13 mammals. NCR3 is a member of the NCR family, which represents the major NK cells triggering receptors. It has been involved in the recognition and killing of tumoral and infected cells, and in the maturation of dendritic cells. By using a combination of nested RT-PCR and RNA-seq analysis, it was possible to detect the expression of several NCR3 transcripts in all the analysed mammalian species, except in Mus musculus, where NCR3 is a pseudogen. It was observed an increase in the number of coding variants in primates, mainly by the internal splicing of exon 2 and the presence of exons 4II and 4III, which are only expressed in higher primates (Hominoidea). In contrast, the diversity of non-coding variants is similar among all analysed species, although there are some conserved ones, which could have a potential regulatory role. The nine human splice variants, six coding and three non-coding, were only detected at high expression levels in immune-related tissues, being the coding A, B y C the most abundant ones. The predicted human protein isoforms are potential transmembrane type I receptors, which extracellular domains are predicted to be Immunoglobulin type V (IgV) or type C (IgC). The interaction of these potential NCR3 extracellular domains with the ligand B7-H6 was analysed using flow citometry assays. Firstly, they were over-expressed in insect cells finding that defective glycosylation avoids binding. In a second approach, it was used a mammalian system detecting only specific binding of IgV domain to B7H6, indicating that B7H6 is not a ligand for IgC containing isoforms. In conclusion, all the presented data suggest a potential role of AS in the NCR3 functions and immune system regulation and evolution.