TUT1 and USB1, the biogenesis enzymes for 2′,3′-cyclic phosphate modification at U6 snRNA 3’ end, have distinct roles in mammalian development [Nanopore]

The 3’ end of most cellular U6 snRNA has a 2′,3′-cyclic phosphate group, a post-transcriptional modification thought to be essential for proper RNA splicing and cellular function. Recent studies have established that the biogenesis of 2′,3′-cyclic phosphate requires two enzymes: first TUT1 adds a poly(U) tail to the 3’ end of U6 snRNA, followed by USB1-catalyzed trimming of the poly(U) tail. Here, we use nine genetically engineered mouse models of Tut1 and Usb1 to show that, contrary to what was previously believed, Usb1 and thus 2′,3′-cyclic phosphate is dispensable for intra-embryonic cell lineages. We find that Tut1, but not Usb1, is essential for the maintenance of the epiblast and neural stem cells. Loss of Tut1 causes defective RNA splicing, triggering massive DNA damage and subsequent cell death. In contrast, loss of Usb1 primarily damages endothelial cells of the extra-embryonic vasculature in the yolk sac by both cell-intrinsic and -extrinsic mechanisms, leading to fetal growth restriction. These results demonstrate that TUT1-catalyzed U6 snRNA 3’ end oligouridylation, rather than 2′,3′-cyclic phosphate as previously thought, is required for proper RNA splicing. Further studies are warranted to elucidate how these different post-transcriptional modifications regulate the function of U6 snRNA in RNA splicing in development and disease.