The opening of Pandora’s box is said to have cursed the ancient Greek world with sickness, death, and innumerable other evils. Thankfully, a modern-day technique called “PANDORA-seq” brings forth new hope for removing these ancient afflictions on our epigenomes by identifying previously undetectable small non-coding RNAs (sncRNAs).
A hopeful handful of researchers led by Qi Chen (University of California, Riverside) knew that high-throughput RNA sequencing protocols failed to detect a significant fraction of modified sncRNAs. Therefore, this expectant epigenetic team combined enzyme treatments (T4 polynucleotide kinase and α-ketoglutarate-dependent hydroxylase) to remove these pesky RNA modifications that block adapter ligation and reverse transcription (respectively). By combining this library preparation method with improved small RNA bioinformatics pipelines (SPORTS1.1), they have unleashed PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), which opens a “Pandora’s box” of sncRNAs.
Let’s open this hopefully unproblematic epigenetic “can of worms” and hear all about the applications of Pandora-seq from Shi and Colleagues.
- PANDORA-seq identifies previously undetected but abundant modified sncRNAs—mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) – when compared with existing sncRNA-seq methods
- Novel sncRNAs exhibit tissue-specific expression (mouse brain, liver, spleen, mature sperm, and sperm heads) and cell-specific expression in mouse and human embryonic stem cells (ESCs) and human cancer cells (HeLa)
- Further exciting findings from PANDORA-seq include:
- microRNAs do not represent the majority sncRNA population in many tissues/cells
- rsRNAs are the most abundant sncRNA in mature sperm (and not tsRNAs), which opens new areas of exploration related to intergenerational effects and transgenerational epigenetic inheritance
- The detection of sncRNAs derived from Y-RNAs supports the ongoing exploration of their biogenesis and function
- PANDORA-seq reveals previously unidentified sncRNA dynamics during the reprogramming of mouse embryonic fibroblasts into induced pluripotent stem cells
- Downregulated tsRNAs and rsRNAs during reprogramming also impacts translation in ESCs (e.g., 28S-1 or a pool of five tsRNAs), thereby supporting a role for novel sncRNAs in cell fate determination
Instead of bringing famine and plague, PANDORA-seq opens a world of previously hidden sncRNAs to the eager epigenetic research field. However, the authors do note some room for improvements, including the exploration of enzyme treatments for additional tsRNA modifications, the development of an all-liquid-based protocol to avoid repeated RNA extraction, and the introduction of optimal conditions to maintain RNA integrity.
“Currently, these small RNAs can be comprehensively profiled by high-throughput methods such as RNA sequencing,” senior author Qi Chen shared. “However, the widely used small RNA sequencing protocols have intrinsic limitations, which prevent certain modified small non-coding RNAs from being detected during RNA sequencing. PANDORA-seq overcomes these limitations.” First author Junchao Shi concludes, “The new method could revolutionize the view of small RNA landscapes.”
For more on how PANDORA-seq has opened a new world of sncRNAs, see Nature Cell Biology, April 2021.