RNA G-quadruplexes regulate mammalian mirtron biogenesis

Mirtrons are a predominant class of noncanonical microRNAs derived from introns through a Drosha-independent, splicing-dependent pathway. Unregulated splicing of introns containing hairpins may adversely impact Dicer/Ago-mediated canonical microRNA biogenesis. However, the mechanism regulating mirtron biogenesis remains poorly understood. We found that the 5' arm of plant mirtrons and invertebrate mirtrons are enriched for uracils; in contrast, the 5' arm of vertebrate mirtrons are enriched for guanines. Further analysis revealed that most of the mammalian mirtrons contain an RNA G-quadruplex (rG4); this was not observed among plant/invertebrate mirtrons. Interestingly, almost all the rG4s in mammalian mirtrons were present in the 5' arm. Predicted rG4s in human mirtrons form a G-quadruplex structure in vitro and rG4 formation in the 5' arm of mirtrons facilitates splicing-mediated biogenesis of mirtrons. Notably, the disruption of rG4s in the 5' arm of mirtrons inhibits splicing and maturation; while mutations outside the rG4-motif do not impact mirtron biogenesis. Our findings support the notion that rG4s at the 5' arm are key regulatory elements in the evolutionary landscape of mammalian mirtrons. This work advances our current understanding of mirtron biogenesis and highlights additional roles for rG4s in small RNA biology.