The removal of introns from pre-RNA is an essential step in gene expression. The majority of introns within the genome are of the major class or U2-type; however, approximately 700 genes in human (less than 0.05%) are of the minor class or U12-type. Defects in the splicing of genes harbouring minor class introns have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as MOPD (microcephalic osteodysplastic primordial dwarfism) or Taybi-Linder syndrome, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Several prominent human cancer genes contain minor class introns, including key components of the MAPK and PI3K mitogenic pathways.
Our genetic, morphological and biochemical characterisation of a zebrafish intestinal mutant, caliban (clbn), revealed the importance of minor class splicing in vertebrate development. In clbn mutants, the absence of a specific minor class spliceosome component, Rnpc3, leads to the accumulation of aberrant minor class splicing particles, a robust and widespread impact on gene expression, impaired digestive organ development and death between 7-10dpf. RNAseq analysis of wildtype and clbn larvae at 72hpf shows that impaired minor class splicing causes intron retention specifically in minor class intron-containing genes and dysregulated expression of genes that are critical to essential cellular processes, including multiple stages of mRNA processing and quality control. Our results indicate that efficient minor class splicing is required to sustain the growth and division of rapidly proliferating cells.
To investigate whether the regulation of minor class splicing impacts on mammalian development, homeostasis and disease, we generated mouse models of Rnpc3 deficiency. We found that Rnpc3 is indispensable for mouse development, and in adulthood it is required to maintain the integrity of multiple tissues and organs, including the epithelium of the entire gastrointestinal tract.
To test the hypothesis that minor class splicing represents a useful, clinically relevant, target for cancer treatment, we examined the impact of impairing minor splicing in a variety of tumour-prone mouse models. Remarkably, we found that Rnpc3 heterozygosity decreases the gastric tumour burden in mice with elevated Stat3 activity and also in Pten heterozygous mice which spontaneously develop large lymphomas. Moreover, homozygous deletion of conditional Rnpc3 alleles by intranasal Adenoviral Cre administration decreased the growth of lung adenocarcinomas caused by the conditional expression of oncogenic K-Ras (K-RasG12D).