Has searching for ncRNAs left you needing forty winks? Maybe take a short rest and dream-a-little-dream, but don’t sleep on a brand new technique known as napRNA sequencing or NAP-seq, which represents a wake-up call to the epigenetics field by providing the ability to describe novel classes of human regulatory non-coding or ncRNAs! Wakey-wakey!
RNA polymerases I-III transcribe the genome into RNAs, which become processed into capped RNAs (mainly messenger RNAs), and non-capped RNAs (napRNAs), which can serve as non-coding RNAs (ncRNAs) to regulate gene expression. To describe the genome-wide prevalence, mechanism, and function of napRNAs, a wide-awake team of researchers led by Bin Li, Lianghu Qu, and Jianhua Yang (Sun Yat-sen University) developed “NAP-seq” to globally profile full-length napRNAs carrying various terminal modifications at the single-nucleotide resolution in the hope of discovering diverse classes of structured ncRNAs.
NAP-seq leverages enzymatic treatments (T4PNK, RNase H, and SuperScript IV), improves adapter ligation and strand-specific/quantitative sequencing, and avoids mispriming artifacts, ribosomal RNA contamination, and difficulties related to RNA modifications/RNA secondary structures during sequencing library construction. Furthermore, comparisons with traditional techniques demonstrate that NAP-seq can accurately detect previously unidentified structured and multiple classes of stably expressed napRNAs in diverse genomic locations
Let’s hear from Liu, Huang, and Colleagues on why you shouldn’t sleep on NAP-seq when exploring ncRNAs:
- NAP-seq of human and mouse napRNA transcriptomes enables the analysis of their evolutionary conservation and the evaluation of their dynamic responses to various stimuli and differentiation stages
- The diverse classes of napRNAs include stably expressed linear intron napRNAs (sliRNAs), a class of snoRNA-intron (snotron) napRNAs, a class of napRNAs embedded in miRNA spacer regions (misRNAs)
- Simultaneously, they detect thousands of previously uncharacterized structured napRNAs and repetitive-element-derived napRNAs (repRNAs)
- The longer half-lives of napRNAs than enhancer RNAs and long non-coding RNAs imply their potential functional roles; however, napRNAs characterized by low abundance, poor conservation, and overlapping interspersed repeated elements may also represent degradation products/RNA intermediates
- NAP-seq helps to identify a long, structured napRNA that regulates myoblast differentiation in mice and a previously undescribed napRNA (DINAP—a composite box C/D-H/ACA RNA) that interacts with dyskerin pseudouridine synthase 1 (DKC1) to maintain protein stability and promote hepatoblastoma cell proliferation
NAP-seq provides a wake-up call to those exploring the ncRNA universe by highlighting the sliRNAs, snotrons, misRNAs, undescribed structured napRNAs, and repRNAs currently “slumbering” in the human genome. The next “dream” for these wide-awake researchers is to classify and elucidate the roles of napRNAs in a wide range of processes.
For more on how NAP-seq has woken up the epigenetics field, see Nature Communication, March 2024.