Full-length Sequencing of Non-capped RNAs Reveals the Heterogeneity of RNA Processing and Novel Classes of Noncoding RNAs

Purpose:Up to 80% of the human genome gives rise to “dark matter” RNAs that mainly composes of non-capped RNAs (napRNAs). However, determining the functional impacts and metabolisms of the napRNAs requires identification of their full-length sequences. Method:We developed an approach to globally profile the full-length sequences of napRNAs at single-nucleotide resolution Results:We discovered, for the first time, the stable expressed linear intron RNA (sliRNA), a novel class of snoRNA-intron (snotron) RNAs, a new class of RNA embedded in miRNA spacers (misRNA) and thousands of new exceptional structured ncRNAs and repeat-derived ncRNAs (repeatRNA) in humans and mouse. Importantly, these new napRNAs are dynamic changes in response to various stimuli and biological processes, suggesting that they have potential regulatory functions. Furthermore, we show that functional napRNA FS4, discovered by NAP-seq firstly, promotes HepG2 cell proliferation by interacting with DKC1 to maintain the protein stability. Conclusion:Collectively, our approach establishes a paradigm for mapping and annotating novel class of ncRNAs of many living species. Next generation sequencing of NAP-seq 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 of NAP-seq and normal RNA-seq was applicated in non-treated HepG2 and C2C12. NAP-seq combined with SHAPE-Map was applicated in non-treated HepG2. Each sample has three biological replicates (except for CoCl2-treated HepG2, which has two).