TRANSGENIC EXPRESSION OF SMALL NONCODING VAULT RNA1-1 DISRUPTS INTESTINAL EPITHELIAL RENEWAL AND CAUSES GUT BARRIER DYSFUNCTION

Small noncoding vault RNAs (vtRNAs) are highly expressed in many eukaryotes and have been linked to different cell functions and pathologies. Humans express four vtRNA paralogs, including vtRNA1-1, vtRNA1-2, vtRNA1-3, and vtRNA2-1, but their biological functions in the intestinal epithelium are underexplored. In this study, we developed transgenic mice that specially overexpress vtRNA1-1 in the intestinal epithelium (vtR1-1Tg) and demonstrate that tissue-specific overexpression of vtRNA1-1 inhibits renewal of the small intestinal mucosa and impairs gut barrier function. Methods: The intestinal epithelial tissue-specific A33-promoter was chosen to generate vtR1-1Tg mice in this study. The cDNA encoding human vtRNA1-1 was cloned to IRES-GFP vector, and the final DNA construct (A33-vtRNA1-1-GFP-NLS) was used for microinjection to embryos. The levels of vtRNAs in gut mucosa and other tissues were measured by qPCR analysis and GFP immunofluorescence assay. Mucosal growth and gut permeability were measured by BrdU incorporation and tracer FITC-dextran assays. Primary enterocytes were isolated from the small intestine of mice for deriving intestinal organoids. Results: Two founders were generated after microinjection and their germ line transmissions were checked by genotyping. vtR1-1Tg mice showed the specific overexpression of vtRNA1-1/GFP in the small intestinal mucosa only, whereas vtRNA1-1 levels were undetectable in the gastric mucosa, liver, kidney, and lung. There were no significant differences in body weight and overall gut mucosal structure between vtR1-1Tg mice and control littermates. However, vtR1-1Tg mice exhibited a significant inhibition of small intestinal mucosal growth, as evidenced by decreased BrdU staining, reduced levels of proliferation-associated proteins Ki67 and PCNA, and shrinkages of crypts and villi in the mucosa of vtR1-1Tg mice relative to littermates. Similarly, intestinal organoids derived from vtR1-1Tg mice grew much slower than those generated from littermate mice. Transgenic expression of vtRNA1-1 also decreased the levels of intercellular junction proteins claudin-7, occludin, JAM-A, ZO-2, and E-cadherin. When challenged with a low dose of LPS (0.15 mg/kg, I.P. injection daily for 5 days), vtR1-1Tg mice showed increased vulnerability of the gut barrier, since gut permeability by LPS in vtR1-1Tg mice was much higher than that observed in littermates. Interestingly, extracellular vesicles isolated from the serum of patients with septic shock exhibited increased levels of vtRNA1-1 and vtRNA2-1, which was associated with gut barrier dysfunction. Conclusions: These results indicate that vtRNA1-1 functions as a negative regulator of intestinal epithelial renewal and gut barrier function, demonstrating the importance of controlling vtRNA1-1 levels in maintaining intestinal epithelial homeostasis.