RNA Polymerase III is Required for Intestinal Epithelial Development and Paneth Cell Maturation in Mice

Background and aims: RNA polymerase III transcribes small non-coding RNAs, indispensable for protein synthesis and cell growth control, especially in fast self-renewing tissues such as the intestinal epithelium. Recently, the importance of the RNA polymerase III complex in digestive development was demonstrated using zebrafish genetics. However, the role of RNA polymerase III in mammalian organisms has not been studied so far. We investigated the effects of disruption of the RNA polymerase III complex on the mouse intestinal epithelium, using tissue-specific gene mutation of the Polr3b gene, encoding the second largest subunit of the RNA polymerase III complex. Medhods: The conditional Polr3b mutant allele, with LoxP sites flanking exon 10 of the Polr3b gene, was derived in mouse embryonic stem cells via homologous recombination, allowing for ablation of exon 10 in Polr3bloxP/loxP upon Cre recombinase activation in Polr3bloxP/loxP; VillinCre mice. Results: Polr3bloxP/ loxP;Villin-Cre mice showed nearly 50% lower survival rate within the first postnatal week and significantly lower body weight. Surviving mutants developed severely disorganized intestinal epithelia, characterized by villus branching, and increased number and expansion of crypts. Furthermore, the intestinal epithelium of Polr3b mutant mice showed a remarkable increase in proliferative crypt regions and disorganized cellular migration, suggesting impaired cellular turnover in mice with disrupted RNA polymerase III complex. Polr3b mutant mice did not display major abnormalities in enterocyte or Goblet cell differentiation. However, Paneth cells in three-week old Polr3bLoxP/LoxP;VillinCre mice were not confined to the crypts, but expanded into the villus region. Surprisingly, mis-positioned Paneth cells resembled Goblet cell by morphology, and expressed both lysozyme and mucin 2, Paneth cell and Goblet cell markers, respectively. Gene profile analysis of newborn Polr3bloxP/ loxP;Villin-Cre mice demonstrated reduced expression of the Paneth cell genetic program. Overall, the strongest phenotype was apparent in three week-old Polr3b mutant mice. After the weaning period, the surviving of Polr3b mutant mice caught up in weight with their littermates; however, increased proliferative activity and disorganization of the intestinal epithelium persisted in adult Polr3bloxP/loxP;Villin-Cre mice. Conclusion: These data show that the RNA polymerase III complex regulates development of the intestinal epithelium and Paneth cell maturation. The RNA polymerase III complex plays an essential role in cell cycle dynamics of the intestinal epithelium inmice. Our future studies will focus on addressing the question whether targeting of RNA polymerase III is feasible for treatment of cancer.