NAP-seq Reveals Novel Classes of Structured Noncoding RNAs with Regulatory Functions

Purpose:Up to 80% of the human genome produces “dark matter” RNAs, most of which are noncapped RNAs (napRNAs). However, determining the functional impacts and metabolism of napRNAs requires the identification of their full-length sequences. Method:Here, by developing a novel method, NAP-seq, to globally profile the full-length sequences of napRNAs at single-nucleotide resolution, we revealed several novel classes of exceptionally structured noncoding RNAs (ncRNAs). Results:We discovered stably expressed linear intron RNAs (sliRNAs), a novel class of snoRNA-intron RNAs (snotrons), a new class of RNAs embedded in miRNA spacers (misRNAs) and thousands of new structured ncRNAs in humans and mice. These new napRNAs undergo dynamic changes in response to various stimuli and differentiation stages. Importantly, we showed that the novel napRNA DINAP interacts with dyskerin pseudouridine synthase 1 (DKC1) to promote cell proliferation by maintaining DKC1 protein stability. Conclusion:Our approach establishes a paradigm for discovering novel classes of ncRNAs with regulatory potency. Next generation sequencing was applicated in 6 human cell types, including U87, HEK293T, HepG2 (including 4 conditions: non-treated, poly(I:C)-treated, Adriamycin-treated, CoCl2-treated), and in 4 differentiation stages of mouse skeletal muscle cell C2C12, including 0 hour, 72 hours, 96 hours, 144 hours after differentiation. Third generation sequencing was applicated in non-treated HepG2. Each sample has three biological replicates (except for CoCl2-treated HepG2, which has two).