RNA METHYLATION SUSTAINS INTESTINAL HOMEOSTASIS AND REGENERATION

The intestinal epithelium performs numerous indispensable functions including absorbing water and nutrients and acting as a barrier against gut bacteria and viruses. Existing literature indicates that intestinal stem cells alter DNA transcription to maintain intestinal epithelial homeostasis and regeneration. Outside of the gut, a variety of adult stem cells require RNA N6-adenomethylation (m6A) for both homeostatic and regenerative processes, but little is known regarding m6A in the intestinal epithelium. Defining a role for m6A in the homeostatic and regenerating intestinal epithelium would establish a new paradigm for investigating gene regulation in this tissue and ultimately produce new opportunities for identifying therapeutic targets in intestinal disease. We deleted METTL3 in the intestinal epithelium of both developing and adult mice and found that METTL3 is essential for the survival of highly proliferative stem progenitor cells known as transit amplifying (TA) cells). TA cell death led to the collapse of mucosal morphology and the loss of absorptive cells, resulting in uniform wasting and death in mice. Mechanistically, METTL3 deletion decreased translational efficiency of hundreds of methylated transcripts as determined by polysome profiling and m6A sequencing. This included downregulated expression of transcripts involved in growth factor signal transduction, including the proto-oncogene and master growth regulator, Kras. We conclude that METTL3 is essential for intestinal homeostasis by promoting translation of growth factor pathway members in transit amplifying cells. Next, we explored the role of m6A during regeneration by performing m6A sequencing in the regenerating intestinal epithelium both in vivo and in vitro. We found that m6A methylates multiple critical pro-regenerative pathways including the adaptive response to hypoxia. Knockdown of METTL3 in colonic organoids (colonoids) had no effect on gross colonoid appearance but led to substantial increases in hypoxic cell death. In wildtype colonoids, hypoxia dramatically induced the m6A binding protein, IMP1, which was also required for survival during hypoxic injury. We conclude that METTL3 is essential for the adaptive pro-survival response to hypoxia in an IMP1-dependent manner. These data demonstrate essential roles for m6A during intestinal homeostasis and regeneration and lay the groundwork for future exploration of this gene regulatory paradigm in the intestinal epithelium.