An evolutionarily conserved function of the pre-mRNA splicing regulator TNPO3 for T cell development

All vertebrates possess lymphocytes, enabling the functional interrogation of this evolutionarily conserved organizing principle of adaptive immunity in non-mammalian species. A forward genetic screen in zebrafish indicated that a mutation of tnpo3, encoding a regulator of alternative splicing of pre-mRNAs, specifically impaired intrathymic T cell development. In mouse T cells, Tnpo3 deficiency predominantly disrupted the splicing of genes encoding RNA-binding motifs, and it specifically blocked the splicing of Va11, associated with reduced numbers of peripheral T cells and the complete loss of iNKT cells. Our results reveal an evolutionarily conserved function of a pre-mRNA processing factor for T cell development, uncovering a previously unappreciated checkpoint during the formation of mature mRNAs of the Tcra gene. Overall design: We identified a loss-of-function mutation in the zebrafish tnpo3 gene. We also generated conditional knock-out mice using a floxed allele of Tnpo3 and Lck:Cre (Orban et al., 1992) to eliminate Tnpo3 in the aß lineage of T cells. Under these conditions, complete inactivation of the Tnpo3 gene was achieved. RNA was prepared from three replicates of wild-type and mutant zebrafish. RNA was also prepared from three replicates of CD4-positive and CD4/CD8-double-positive thymocytes of heterozygous (Tnpo3fl/+; Lck:Cre) and homozygous (Tnpo3fl/fl; Lck:Cre) mutant mice. RNAs were subjected to RNA-seq analysis and examined for differentially expressed genes and alternative splicing events.