PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications

Abstract

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.RNA modifications, warrant future extensive investigations in different systems.

MLC Seq: De novo sequencing of full-length tRNA isoforms by mass ladder complementation

“…To differentiate different methylations in the tRNA-Glu, we included an additional step in the MLC Seq workflow by treating the tRNA-Glu sample with AlkB, which is a known enzyme that can demethylate tRNA-rich modifications such as 3-methylcytosine (m 3 C), 1-methyladenosine (m 1 A), and methylguanosine (m 1 G) ( Fig. S2 ) 8 .…”

“…The enriched tRNA-Glu was also validated by the Northern Blot. The gel recovery process was described in our previous paper 7 . The probe was synthesized by IDT and the sequence was listed below: tRNA-Glu pulldown probe: 5’-Biotin-CTAACCACTAGACCACCAGGGA.…”

“…It is also challenging for current next generation sequencing (NGS) methods to sequence them due to cDNA synthesis-blocking modifications. There are methods/enzymes used to remove these cDNA-blocking modifications 8 that allow the tRNA’s primary sequences to be read, but at the expense of losing the modification information. Nanopore RNA-Seq has been used recently for sequencing of tRNAs 9 .…”

Regulation of Ribosome Function by RNA Modifications in Hematopoietic Development and Leukemia: It Is Not Only a Matter of m6A

“…More recently, a novel high-throughput RNA sequencing methodology referred to as panoramic RNA display by overcoming RNA modification aborted sequencing (PANDORA-seq) has enabled the identification of ribosomal RNA-derived small RNA fragments (rsRNAs) [ 69 ]. Notably, rsRNAs present tissue- and cell-specific expression, thus indicating a possible role in cell identity.…”

“…Notably, rsRNAs present tissue- and cell-specific expression, thus indicating a possible role in cell identity. Moreover, the transfection of a 28S rRNA-derived rsRNA in mouse embryonic stem cells (mESCs) significantly reduced translation [ 69 ]. Thus, the impact of rRNA modifications on rsRNA biogenesis and their potential regulatory roles in normal and malignant hematopoiesis warrant further investigation.…”