Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing (PANDORA) is a new RNA sequencing method developed by scientists from the University of California, Riverside (UCR), and published in the journal Nature Cell Biology in a paper titled “PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications.”

Pandora-seq uses combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription allowing the identification of abundant modified sncRNAs, mainly tsRNA and rsRNA, which previously could not be easily detected (Fig. 1).

TsRNA (small tRNA-derived RNA) and rsRNA (small rRNA-derived RNA) originate from the cleavage of tRNA and rRNA and exist in all domains of life, including archaea, bacteria and eukaryotes. The presence and relatively high abundance of tsRNA and rsRNA has been highlighted in many high-throughput sequencing datasets, thus suggesting their important role as biologically active entities. In fact, tsRNA and rsRNA are present in living organisms under physiological conditions and are involved in the response to environmental stress and in various processes such as transcriptional regulation, control of retrotransposons, epigenetic inheritance.

The complex RNA modification scenarios leading to the synthesis of these small RNAs have caused problems in high-throughput assays as they interfere with the preparation of the RNA-seq library and prevent the detection of tsRNA and rsRNA bearing certain modifications. This new RNA sequencing technique, according to the researchers who developed it, would instead allow to detect even these small RNAs that originated as a result of these complex modifications, solving the detection problems by improving, as previously mentioned, both the binding of the adapter that performs reverse transcription during the construction of the RNA-seq library thus allowing the detection of a much larger number of small RNAs.

Figure 1. a, Schematics of the RNA properties (terminal and internal modifications) and key steps (adapter ligation and reverse transcription) of traditional RNA-seq, AlkB-facilitated RNA-seq, T4PNK-facilitated RNA-seq and PANDORA-seq. b, Schematic of the detection capacities of the abovementioned RNA-seq protocols from a small RNA pool. c, Demethylation activity of m1A, m1G, m3C and m22G with or without AlkB treatment of 15- to 50-nucleotide RNA fractions from mouse tissue (liver), as revealed by LC-MS/MS (n = 3 biologically independent samples). The data represent means ± s.e.m. Statistical significance was determined by two-sided multiple t-test (**P < 0.01; ***P < 0.001). d, Validation of improvements in 3′ terminal ligation following T4PNK treatment in synthesized tsRNAs and small RNA fractions extracted from mouse tissue (spleen). nt, nucleotides. e, Northern blot analysis of the 3′ adapter sequence to show, semi-quantitatively, improvement in the number of adapters being ligated before and after treatment with T4PNK. f–i, The improved treatment protocol minimized the potential artificial increase in tsRNAs and rsRNAs due to de novo degradation of tRNAs and rRNAs. In f and g, AlkB treatment on total RNAs (from HeLa cells) resulted in increased tsRNA (f) and rsRNA products (g), as observed by increased RNA smear (left) and by northern blots (right). In h and i, northern blot analyses of tsRNAs (h) and rsRNAs (i) after AlkB and/or T4PNK treatment on pre-size-selected RNA fractions (15- to 50-nucleotide RNA from HeLa cells) did not result in further degradation. For d–i, similar results were obtained in three independent experiments. j, Comparison of the PANDORA-seq results using treatment with either T4PNK first and AlkB second (T4PNK + AlkB) or AlkB first and T4PNK second (AlkB + T4PNK) in HeLa cells (15- to 50-nucletide RNA) showed highly consistent results (Spearman’s correlation; ρ = 0.995). Correlation coefficients for comparisons between other protocols are also provided. Statistical source data, precise P values and unprocessed blots are provided in the source data.
Source: https://www.nature.com/articles/s41556-021-00652-7

To learn more about the topic, I invite you to read the article that these researchers published in Nature to get into the details of the technique and understand how it works. You can directly access the article by clicking here.

As Tong Zhou, a bioinformatician at the University of Nevada, Reno School of Medicine and co-author of the study, said, "PANDORA-seq has opened Pandora's box of small RNAs"

Sources:

  • Shi, J., Zhang, Y., Tan, D. et al. PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications. Nat Cell Biol 23, 424–436 (2021); https://doi.org/10.1038/s41556-021-00652-7
  • Oberbauer, V., & Schaefer, M. R. (2018). tRNA-Derived Small RNAs: Biogenesis, Modification, Function and Potential Impact on Human Disease Development. Genes, 9(12), 607; https://doi.org/10.3390/genes9120607