Zhang, Liwen, Ph.D.

Small noncoding RNAs (sncRNAs) are a diverse group of RNAs including small interfering RNAs (siRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs) and RNAs derived from structured RNAs like transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and others. These sncRNAs have varied termini and RNA modifications, which can interfere with adaptor ligation and reverse transcription during cDNA library construction, hindering detection of many types of sncRNAs by standard small RNA sequencing methods. To address this limitation, PANDORA-seq introduces a refined methodology. The procedure includes sequential enzymatic treatments of size-selected RNAs with T4PNK and AlkB, which effectively circumvent the challenges presented by the ligation-blocking termini and reverse transcription-blocking RNA modifications, followed by tailored small RNA library construction protocols and deep sequencing. The obtained datasets are analyzed with the SPORTS pipeline, which can comprehensively analyze various types of sncRNAs beyond the traditionally studied classes, to include those derived from various parental RNAs (e.g., from tRNA and rRNA), as well as output the locations on the parental RNA from which these sncRNAs are derived. The entire protocol takes  7 days, depending on the sample size and sequencing turnaround time. PANDORA-seq provides a transformative tool to further our understanding of the expanding small RNA universe and to explore the uncharted functions of sncRNAs.

Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs-mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)-that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation.