Researchers from Sun Yat-sen University have developed a new method called NAP-seq that shines light on the hidden world of “dark matter” RNAs. These RNAs, which make up nearly 80% of the human genome, often go unnoticed because they lack the chemical cap usually found at the start of RNA molecules. Despite this, they play important roles in regulating how genes work.

Using NAP-seq, the team was able to capture the full-length sequences of these unusual RNAs at single-nucleotide resolution. They discovered several new categories of noncoding RNAs including stably expressed linear intron RNAs (sliRNAs), snoRNA-intron RNAs (snotrons), and RNAs located in microRNA spacer regions (misRNAs). In addition, they identified thousands of previously unknown structured RNAs in both humans and mice.

NAP-seq accurately identifies full-length napRNAs at single-nucleotide resolution

a Schematic depiction of the NAP-seq method with the construction of TGS libraries and NGS libraries. Total RNA was extracted and pre-size selection of RNA fractions was performed by removing short RNAs. b The percentage of reads with at least one specific adapter in the NAP-seq-NGS libraries in humans and mice (3 biological replicates per sample). All data are plotted as the means ± SEMs. The number of reads (RPM, reads per million) for known ncRNAs was highly reproducible between replicates with both the NAP-seq-NGS (c) and NAP-seq-TGS (d) methods. p values were calculated by two-sided Pearson’s correlation test. e, f Coincidence analysis of both terminal sites in the NAP-seq-NGS reads and those in the known ncRNAs in HepG2 cells. The x-axis shows the distance from the annotated 5’-start (e) or 3’-end (f) site in the NAP-seq-NGS read to the corresponding terminal sites in the known ncRNAs, and the y-axis shows the number of reads (RPM) within a certain distance from the 5’-start or 3’-end site. The bottom panel shows a heatmap, in which each row represents a gene that matches the terminal sites with the NAP-seq-NGS read exactly, and each column represents the expression values of genes at a specific distance. expr, expression value. g Number of coincidences between the 5’-start sites in NAP-seq-TGS reads and the 5’-start sites in known ncRNAs in HepG2 cells. h Number of coincidences between the 3’-end sites in NAP-seq-TGS reads and the 3’-end sites in known ncRNAs in HepG2 cells. i Genome Browser visualization of NAP-seq 5’-start, 3’-end and coverage signals (RPM, reads per million), which were generated from NAP-seq-NGS and NAP-seq-TGS data, in a 5000-bp region of a human snoRNA cluster (chr17:7,573,750-7,578,750).

These RNAs are not just passive byproducts. The researchers found that they change dynamically during different stages of cell growth and in response to environmental signals. Importantly, they showed that some of these RNAs actively regulate key biological processes. For example, one structured napRNA was found to control muscle cell differentiation, while another, called DINAP, helps stabilize an important protein, DKC1, to promote cell proliferation.

This work highlights how much remains to be learned about RNA biology and demonstrates that advanced sequencing methods such as NAP-seq can uncover powerful new regulators hidden within the genome.